Methods and means for treatment of osteoarthritis

ABSTRACT

The invention relates to the field of medicinal research, cartilage physiology and diseases involving the degeneration of cartilage tissue. More specifically, the invention relates to methods and means for identifying compounds that inhibit catabolic processes in chondrocytes and that decrease the degradation of cartilage and/or ECM. The invention also relates to the compounds that are useful in the treatment of osteoarthritis. The invention also relates to targets, the modulation of which results in a decrease in the degradation of ECM and/or cartilage and decrease inflammation. In addition, the invention relates to compositions and methods for the use thereof in treating conditions that are characterized by the degradation of ECM and/or cartilage and inflammation.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a Divisional Application of U.S. applicationSer. No. 14/162,908 filed Jan. 24, 2014, now abandoned, which is adivisional of National Stage Application U.S. Ser. No. 13/262,634 filedOct. 1, 2011, now U.S. Pat. No. 8,663,930, claiming the priority ofco-pending PCT Application No. PCT/EP2010/054412 filed Apr. 1, 2010,which in turn, claims priority from U.S. Provisional Application Ser.No. 61/211,740 filed Apr. 1, 2009. Applicants claim the benefits of 35U.S.C. § 120 as to the U.S. Application and U.S. National Stageapplication and the PCT application and priority under 35 U.S.C. § 119as to the said U.S. provisional application, and the entire disclosuresof both applications are incorporated herein by reference in theirentireties.

FIELD OF THE INVENTION

The invention relates to the field of medicinal research, inflammation,cartilage physiology and diseases involving inflammation and/or thedegeneration of cartilage tissue and/or extracellular matrix (ECM). Morespecifically, the present invention relates to agents, and methods foridentifying compounds, which agents and compounds inhibit catabolicprocesses in chondrocytes and that would decrease the degradation ofcartilage and/or extracellular matrix. The invention also relates totargets, the modulation of which results in a decrease in thedegradation of ECM and/or cartilage and decrease inflammation. Inaddition, the invention relates to compositions and methods for the usethereof in treating conditions that are characterized by the degradationof ECM and/or cartilage and inflammation. The invention also relates tothe compounds that are useful in the treatment of osteoarthritis.

BACKGROUND OF THE INVENTION

Cartilage is an avascular tissue of which chondrocytes are the maincellular component. The chondrocytes in normal articular cartilageoccupy approximately 5% of the tissue volume, while the extra-cellularmatrix makes up the remaining 95% of the tissue. The chondrocytessecrete the components of the matrix, mainly proteoglycans andcollagens, which in turn supply the chondrocytes with an environmentsuitable for their survival under mechanical stress. In cartilage,collagen type II, together with the protein collagen type IX, isarranged in solid fibril-like structures which provide cartilage withgreat mechanical strength. The proteoglycans can absorb water and areresponsible for the resilient and shock absorbing properties of thecartilage.

One of the functional roles of cartilage in the joint is to allow bonesto articulate on each other smoothly. Loss of articular cartilage,therefore, causes the bones to rub against each other leading to painand loss of mobility. The degradation of cartilage can have variouscauses. In inflammatory arthritides, as rheumatoid arthritis forexample, cartilage degradation is caused by the secretion of proteases(e.g. collagenases) by inflamed tissues (the inflamed synovium forexample). Cartilage degradation can also be the result of an injury ofthe cartilage, due to an accident or surgery, or exaggerated loading or‘wear and tear’. Cartilage degradation may also be the result of animbalance in cartilage synthesizing (anabolic) and cartilage degrading(catabolic) processes. The ability of cartilage tissue to regenerateafter such insults is limited. Chondrocytes in injured cartilage oftendisplay reduced anabolic activity and/or increased catabolic activity.The limited ability of cartilage to self-repair after injury, disease,or surgery is a major limiting factor in rehabilitation of degradingjoint surfaces and injury to meniscal cartilage.

The degeneration of cartilage is the hallmark of various diseases, amongwhich rheumatoid arthritis and osteoarthritis are the most prominent.

Osteoarthritis (also referred to as OA, or wear-and-tear arthritis) isthe most common form of arthritis and is characterized by loss ofarticular cartilage, often associated with hypertrophy of the bone andpain. The disease mainly affects hands and weight-bearing joints such asknees, hips and spines. This process thins the cartilage. When thesurface area has disappeared due to the thinning, a grade Iosteoarthritis is reached; when the tangential surface area hasdisappeared, grade II osteoarthritis is reached. There are furtherlevels of degeneration and destruction, which affect the deep and thecalcified cartilage layers that border with the subchondral bone. For anextensive review on osteoarthritis, we refer to Wieland et al., 2005.

Rheumatoid arthritis (RA) is a chronic joint degenerative disease,characterized by inflammation and destruction of the joint structures.When the disease is unchecked, it leads to substantial disability andpain due to loss of joint functionality and even premature death. Theaim of an RA therapy, therefore, is not to slow down the disease but toattain remission in order to stop the joint destruction. Besides theseverity of the disease outcome, the high prevalence of RA (˜0.8% of theadults are affected worldwide) means a high socio-economic impact. (Forreviews on RA, we refer to Smolen and Steiner (2003); Lee and Weinblatt(2001); Choy and Panayi (2001); O'Dell (2004) and Firestein (2003)).

The clinical manifestations of the development of the osteoarthritiscondition are: increased volume of the joint, pain, crepitation andfunctional disability that lead to pain and reduced mobility of thejoints. When disease further develops, pain at rest emerges. If thecondition persists without correction and/or therapy, the joint isdestroyed leading to disability. Replacement surgery with totalprosthesis is then required.

In mature articular cartilage, chondrocytes maintain thecartilage-specific matrix phenotype. Early signs of OA includeprogressive loss from articular cartilage of the proteoglycan aggrecan,due to damage to type II collagen. This protein represents the majorstructural collagen found in articular cartilage in healthy individuals.There is ordinarily a strict balance between the production of type IIcollagen and degradation of this protein by catabolic enzymes duringnormal remodeling of cartilage. Pathological conditions such as OA arecharacterized by a loss of this balance with increased proteolysis.

In general, elevated expression of MMPs is associated with thedegradation of cartilage and/or extracellular matrix (ECM) but not allproteases are capable of degrading native collagen. Among the matrixmetallo proteinases, MMP1, MMP8, MMP13 and MMP14 display the highestcapacity for degrading collagen type II. Expression and contents ofMMP-1 (collagenase-1) and MMP-13 (Mitchel et al., 1996; Shlopov et al.,1997), expression of MMP-8 (collagenase-2), and collagenase activity(Billinghurst et al., 1997, Dahlberg et al., 2000) are upregulated inhuman OA cartilage. In particular, MMP-13, also known as collagenase-3,is thought to play an important role in type II collagen degradation inarticular cartilage and especially in OA (Billinghurst et al., 1997,Mitchell et al., 1996, Dahlberg et al., 2000, Billinghurst et al., 2000)as indicated by various observations. 1) The expression of MMP13 isincreased in the cartilage of OA patients and of animals subjected toarthritogenic surgery like meniscectomy (Appleton et al., 2007) 2) Thelocalization of MMP1 and MMP13 in arthritic cartilage appear to coincidewith the location of cartilage destruction, as revealed by antibodiesrevealing neo-epitopes induced by cartilage cleavage. (Wu et al., 2002)3) Overexpression of MMP13 in cartilage of transgenic mice lead to anOA-like cartilage destruction phenotype (Neuhold et al., 2001). 4) TypeII collagen is the preferred substrate for MMP-13 (Billinghurst et al.,1997; Mitchell et al., 1996). Taken together, MMP13 is well-accepted asa key player in OA-induced cartilage and ECM degeneration.

Therapeutic methods for the correction of the articular cartilagelesions that appear during osteoarthritic disease have been developed,but so far none of them have been able to mediate the regeneration ofarticular cartilage in situ and in vivo.

Osteoarthritis is difficult to treat. At present, no cure is availableand treatment focuses on relieving pain and preventing the affectedjoint from becoming deformed. Common treatments include the use ofnon-steroidal anti-inflammatory drugs (NSAIDs). Although dietarysupplements such as chondroitin and glucosamine sulphate have beenadvocated as safe and effective options for the treatment oramelioration of osteoarthritis, a recent clinical trial revealed thatboth treatments did not reduce pain associated with osteoarthritis(Clegg et al., 2006). Taken together, no disease modifyingosteoarthritic drugs are available.

In severe cases, joint replacement may be necessary. This is especiallytrue for hips and knees. If a joint is extremely painful and cannot bereplaced, it may be fused. This procedure stops the pain, but results inthe permanent loss of joint function, making walking and bendingdifficult.

Another possible treatment is the transplantation of cultured autologouschondrocytes. Here, chondral cellular material is taken from thepatient, sent to a laboratory where it is expanded. The material is thenimplanted in the damaged tissues to cover the tissue's defects.

Another treatment includes the intra-articular instillation of Hylan G-F20 (e.g. Synvisc®, Hyalgan®, Artz®), a substance that improvestemporarily the rheology of the synovial fluid, producing an almostimmediate sensation of free movement and a marked reduction of pain.

Other reported methods include application of tendinous, periosteal,fascial, muscular or perichondral grafts; implantation of fibrin orcultured chondrocytes; implantation of synthetic matrices, such ascollagen, carbon fiber; administration of electromagnetic fields. All ofthese have reported minimal and incomplete effects, resulting in a poorquality tissue that can neither support the weighted load nor allow therestoration of an articular function with normal movement.

Stimulation of the anabolic processes, blocking catabolic processes, ora combination of these two, may result in stabilization of thecartilage, and perhaps even reversion of the damage, and thereforeprevent further progression of the disease.

The present invention relates to the relationship between the functionof selected proteins identified by the present inventors (hereinafterreferred to as “TARGETS”) and inhibition of cartilage and/orextra-cellular matrix (ECM) degradation and inhibition of inflammation.

SUMMARY OF THE INVENTION

The present invention relates to a method for identifying compounds thatreduce extra-cellular matrix (ECM) and/or cartilage degradationprocesses comprising contacting the compound with a polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 22-42 (hereinafter “TARGETS”), or a functional fragmentthereof, under conditions that allow said polypeptide to bind to thecompound, and measuring a compound-polypeptide property related to theinhibition of ECM and/or cartilage degradation. In a particularembodiment the compound-polypeptide property is the level ofinflammatory cytokines for example IL-1b, IL-6, IL-8, IL-11, TNFα and/orLIF. In a specific embodiment the compound-polypeptide property measuredis expression levels of cartilage degradation proteins or proteases suchas collagenase. In a particular embodiment the compound-polypeptideproperty measured is the expression levels of MMP1, MMP3, MMP8, MMP13,MMP14, and/or ADAMTS4. In a specific embodiment the compound-polypeptideproperty measured is MMP13 expression levels.

Aspects of the present method include the in vitro assay of compoundsusing the polypeptide corresponding to a TARGET, or fragments thereof,such fragments being fragments of the amino acid sequences described bySEQ ID NO: 22-42, and cellular assays wherein TARGET inhibition isfollowed by observing indicators of efficacy including, for example,TARGET expression levels, TARGET enzymatic activity and/or MMP13 levels.

The present invention also relates to

-   -   (1) expression inhibitory agents comprising a polynucleotide        selected from the group of an antisense polynucleotide, a        ribozyme, and a small interfering RNA (siRNA), wherein said        polynucleotide comprises a nucleic acid sequence complementary        to, or engineered from, a naturally occurring polynucleotide        sequence encoding a TARGET polypeptide said polynucleotide        sequence comprising a sequence selected from the group        consisting of SEQ ID NO: 22-42, and    -   (2) pharmaceutical compositions comprising said agent(s), useful        in the treatment, or prevention, of diseases characterized by        ECM degradation and/or cartilage degradation such as        osteoarthritis.

Another aspect of the invention is a method of treatment, or prevention,of a condition related to cartilage and/or ECM degeneration, bone and/orjoint degradation, in a subject suffering or susceptible thereto, byadministering a pharmaceutical composition comprising an effectiveTARGET-expression inhibiting amount of a expression-inhibitory agent oran effective TARGET activity inhibiting amount of a activity-inhibitoryagent. In a particular embodiment the condition is osteoarthritis.

Another aspect of the invention is a method of treatment, or prevention,of a condition related to inflammation, in a subject suffering orsusceptible thereto, by administering a pharmaceutical compositioncomprising an effective TARGET-expression inhibiting amount of aexpression-inhibitory agent or an effective TARGET activity inhibitingamount of a activity-inhibitory agent.

A further aspect of the present invention is a method for diagnosis of acondition related to cartilage and/or ECM degeneration comprisingmeasurement of indicators of levels of TARGET expression and/or activityin a subject.

A further aspect of the present invention is a method for diagnosis of acondition related to inflammation comprising measurement of indicatorsof levels of TARGET expression and/or activity in a subject.

Another aspect of this invention relates to the use of agents whichinhibit a TARGET as disclosed herein in a therapeutic method, apharmaceutical composition, and the manufacture of such composition,useful for the treatment of a disease involving cartilage and/or ECMdegeneration. In particular, the present method relates to the use ofthe agents which inhibit a TARGET in the treatment of a diseasecharacterized by joint degradation, and in particular, a diseasecharacterized by abnormal MMP13 expression. The agents are useful foramelioration or treatment of a disease involving cartilage degradation,including but not limited to osteoarthritis, rheumatoid arthritis,psoriatic arthritis, juvenile rheumatoid arthritis, gouty arthritis,septic or infectious arthritis, reactive arthritis, reflex sympatheticdystrophy, algodystrophy, Tietze syndrome or costal chondritis,fibromyalgia, osteochondritis, neurogenic or neuropathic arthritis,arthropathy, endemic forms of arthritis like osteoarthritis deformansendemica, Mseleni disease, and Handigodu disease; degeneration resultingfrom fibromyalgia, systemic lupus erythematosus, scleroderma, ankylosingspondylitis, congenital cartilage malformations, including hereditarychondrolysis, chondrodysplasias and pseudoachondrodysplasias, andcongenital cartilage malformation related diseases for example microtia,anotia, and metaphyseal chondrodysplasia. In a particular embodiment thedisease is selected from osteoarthritis, rheumatoid arthritis, andinflammatory arthritis. In a particular embodiment the disease isosteoarthritis.

Another aspect of this invention relates to the use of agents whichinhibit a TARGET as disclosed herein in a therapeutic method, apharmaceutical composition, and the manufacture of such composition,useful for the treatment of a disease involving inflammation, includingbut not limited to allergic airways disease (e.g. asthma, rhinitis),autoimmune diseases, transplant rejection, Crohn's disease, rheumatoidarthritis, psoriasis, juvenile idiopathic arthritis, colitis, andinflammatory bowel diseases.

In a further aspect the present invention also relates to methods forthe in vitro production of cartilage tissue.

Another further aspect of the present invention is a pharmaceuticalcomposition comprising a therapeutically effective cartilage and/or ECMdegradation-inhibiting amount of a TARGET inhibitor or itspharmaceutically acceptable salt, hydrate, solvate, or prodrug thereofin admixture with a pharmaceutically acceptable carrier. The presentpolynucleotides and TARGET inhibitor compounds are also useful for themanufacturing of a medicament for the treatment of conditions involvingECM degradation, cartilage degradation, and/or inflammation.

Furthermore, the invention also relates to diagnostic methods.

Other objects and advantages will become apparent from a considerationof the ensuing description taken in conjunction with the followingillustrative drawings

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Shows the principle of the general screening protocol for theMMP13 assay in normal human articular chondrocytes (NHACs)

FIG. 2: Schematic representation of the MMP13 assay in normal humanarticular chondrocyes

FIG. 3: A representative example of the performance of the control platetested with the screening protocol described for the MMP13 assay innormal human articular chondrocytes

FIG. 4: Example of the data obtained during one of the screening batchesof the SilenceSelect® collection

FIG. 5A: An exemplary layout for 3 MOI rescreen runs

FIG. 5B: Example of the results obtained in a 3 MOI rescreen

FIG. 6: Scheme indicating the attrition obtained during screening andthe selection of the hits analysed in more detail.

FIG. 7 lists the oligonucleotide primers used for SYBR® Greenquantitative real-time PCR.

DETAILED DESCRIPTION

The following terms are intended to have the meanings presentedtherewith below and are useful in understanding the description andintended scope of the present invention.

The term ‘agent’ means any molecule, including polypeptides, antibodies,polynucleotides, chemical compounds and small molecules. In particularthe term agent includes compounds such as test compounds or drugcandidate compounds.

The term ‘agonist’ refers to a ligand that stimulates the receptor theligand binds to in the broadest sense.

As used herein, the term ‘antagonist’ is used to describe a compoundthat does not provoke a biological response itself upon binding to areceptor, but blocks or dampens agonist-mediated responses, or preventsor reduces agonist binding and, thereby, agonist-mediated responses.

The term ‘assay’ means any process used to measure a specific propertyof an agent. A ‘screening assay’ means a process used to characterize orselect agents based upon their activity from a collection of agents.

The term ‘binding affinity’ is a property that describes how stronglytwo or more compounds associate with each other in a non-covalentrelationship. Binding affinities can be characterized qualitatively(such as ‘strong’, ‘weak’, ‘high’, or ‘low’) or quantitatively (such asmeasuring the K_(D)).

The term ‘carrier’ means a non-toxic material used in the formulation ofpharmaceutical compositions to provide a medium, bulk and/or useableform to a pharmaceutical composition. A carrier may comprise one or moreof such materials such as an excipient, stabilizer, or an aqueous pHbuffered solution. Examples of physiologically acceptable carriersinclude aqueous or solid buffer ingredients including phosphate,citrate, and other organic acids; antioxidants including ascorbic acid;low molecular weight (less than about 10 residues) polypeptide;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.

The term ‘complex’ means the entity created when two or more compoundsbind to, contact, or associate with each other.

The term ‘compound’ is used herein in the context of a ‘test compound’or a ‘drug candidate compound’ described in connection with the assaysof the present invention. As such, these compounds comprise organic orinorganic compounds, derived synthetically, recombinantly, or fromnatural sources.

The compounds include inorganic or organic compounds such aspolynucleotides, lipids or hormone analogs. Other biopolymeric organictest compounds include peptides comprising from about 2 to about 40amino acids and larger polypeptides comprising from about 40 to about500 amino acids, including polypeptide ligands, enzymes, receptors,channels, antibodies or antibody conjugates.

The term ‘condition’ or ‘disease’ means the overt presentation ofsymptoms (i.e., illness) or the manifestation of abnormal clinicalindicators (for example, biochemical indicators or diagnosticindicators). Alternatively, the term ‘disease’ refers to a genetic orenvironmental risk of or propensity for developing such symptoms orabnormal clinical indicators.

The term ‘contact’ or ‘contacting’ means bringing at least two moietiestogether, whether in an in vitro system or an in vivo system.

The term ‘derivatives of a polypeptide’ relates to those peptides,oligopeptides, polypeptides, proteins and enzymes that comprise astretch of contiguous amino acid residues of the polypeptide and thatretain a biological activity of the protein, for example, polypeptidesthat have amino acid mutations compared to the amino acid sequence of anaturally-occurring form of the polypeptide. A derivative may furthercomprise additional naturally occurring, altered, glycosylated, acylatedor non-naturally occurring amino acid residues compared to the aminoacid sequence of a naturally occurring form of the polypeptide. It mayalso contain one or more non-amino acid substituents, or heterologousamino acid substituents, compared to the amino acid sequence of anaturally occurring form of the polypeptide, for example a reportermolecule or other ligand, covalently or non-covalently bound to theamino acid sequence.

The term ‘derivatives of a polynucleotide’ relates to DNA-molecules,RNA-molecules, and oligonucleotides that comprise a stretch of nucleicacid residues of the polynucleotide, for example, polynucleotides thatmay have nucleic acid mutations as compared to the nucleic acid sequenceof a naturally occurring form of the polynucleotide. A derivative mayfurther comprise nucleic acids with modified backbones such as PNA,polysiloxane, and 2′-O-(2-methoxy) ethyl-phosphorothioate, non-naturallyoccurring nucleic acid residues, or one or more nucleic acidsubstituents, such as methyl-, thio-, sulphate, benzoyl-, phenyl-,amino-, propyl-, chloro-, and methanocarbanucleosides, or a reportermolecule to facilitate its detection.

The term ‘endogenous’ shall mean a material that a mammal naturallyproduces. Endogenous in reference to the term ‘protease’, ‘kinase’,‘factor’, or ‘receptor’ shall mean that which is naturally produced by amammal (for example, and not limitation, a human). In contrast, the termnon-endogenous in this context shall mean that which is not naturallyproduced by a mammal (for example, and not limitation, a human). Bothterms can be utilized to describe both in vivo and in vitro systems. Forexample, and without limitation, in a screening approach, the endogenousor non-endogenous TARGET may be in reference to an in vitro screeningsystem. As a further example and not limitation, where the genome of amammal has been manipulated to include a non-endogenous TARGET,screening of a candidate compound by means of an in vivo system isviable.

The term ‘expressible nucleic acid’ means a nucleic acid coding for aproteinaceous molecule, an RNA molecule, or a DNA molecule.

The term ‘expression’ comprises both endogenous expression andoverexpression by transduction.

The term ‘expression inhibitory agent’ means a polynucleotide designedto interfere selectively with the transcription, translation and/orexpression of a specific polypeptide or protein normally expressedwithin a cell. More particularly, ‘expression inhibitory agent’comprises a DNA or RNA molecule that contains a nucleotide sequenceidentical to or complementary to at least about 15-30, particularly atleast 17, sequential nucleotides within the polyribonucleotide sequencecoding for a specific polypeptide or protein. Exemplary expressioninhibitory molecules include ribozymes, double stranded siRNA molecules,self-complementary single-stranded siRNA molecules (shRNA), geneticantisense constructs, and synthetic RNA antisense molecules withmodified stabilized backbones.

The term ‘fragment of a polynucleotide’ relates to oligonucleotides thatcomprise a stretch of contiguous nucleic acid residues that exhibitsubstantially a similar, but not necessarily identical, activity as thecomplete sequence. In a particular aspect, ‘fragment’ may refer to aoligonucleotide comprising a nucleic acid sequence of at least 5 nucleicacid residues (preferably, at least 10 nucleic acid residues, at least15 nucleic acid residues, at least 20 nucleic acid residues, at least 25nucleic acid residues, at least 40 nucleic acid residues, at least 50nucleic acid residues, at least 60 nucleic residues, at least 70 nucleicacid residues, at least 80 nucleic acid residues, at least 90 nucleicacid residues, at least 100 nucleic acid residues, at least 125 nucleicacid residues, at least 150 nucleic acid residues, at least 175 nucleicacid residues, at least 200 nucleic acid residues, or at least 250nucleic acid residues) of the nucleic acid sequence of said completesequence.

The term ‘fragment of a polypeptide’ relates to peptides, oligopeptides,polypeptides, proteins, monomers, subunits and enzymes that comprise astretch of contiguous amino acid residues, and exhibit substantially asimilar, but not necessarily identical, functional or expressionactivity as the complete sequence. In a particular aspect, ‘fragment’may refer to a peptide or polypeptide comprising an amino acid sequenceof at least 5 amino acid residues (preferably, at least 10 amino acidresidues, at least 15 amino acid residues, at least 20 amino acidresidues, at least 25 amino acid residues, at least 40 amino acidresidues, at least 50 amino acid residues, at least 60 amino residues,at least 70 amino acid residues, at least 80 amino acid residues, atleast 90 amino acid residues, at least 100 amino acid residues, at least125 amino acid residues, at least 150 amino acid residues, at least 175amino acid residues, at least 200 amino acid residues, or at least 250amino acid residues) of the amino acid sequence of said completesequence.

The term ‘hybridization’ means any process by which a strand of nucleicacid binds with a complementary strand through base pairing. The term‘hybridization complex’ refers to a complex formed between two nucleicacid sequences by virtue of the formation of hydrogen bonds betweencomplementary bases. A hybridization complex may be formed in solution(for example, C_(0t) or R_(0t) analysis) or formed between one nucleicacid sequence present in solution and another nucleic acid sequenceimmobilized on a solid support (for example, paper, membranes, filters,chips, pins or glass slides, or any other appropriate substrate to whichcells or their nucleic acids have been fixed). The term “stringentconditions” refers to conditions that permit hybridization betweenpolynucleotides and the claimed polynucleotides. Stringent conditionscan be defined by salt concentration, the concentration of organicsolvent, for example, formamide, temperature, and other conditions wellknown in the art. In particular, reducing the concentration of salt,increasing the concentration of formamide, or raising the hybridizationtemperature can increase stringency. The term ‘standard hybridizationconditions’ refers to salt and temperature conditions substantiallyequivalent to 5×SSC and 65° C. for both hybridization and wash. However,one skilled in the art will appreciate that such ‘standard hybridizationconditions’ are dependent on particular conditions including theconcentration of sodium and magnesium in the buffer, nucleotide sequencelength and concentration, percent mismatch, percent formamide, and thelike. Also important in the determination of “standard hybridizationconditions” is whether the two sequences hybridizing are RNA-RNA,DNA-DNA or RNA-DNA. Such standard hybridization conditions are easilydetermined by one skilled in the art according to well known formulae,wherein hybridization is typically 10-20^(N)C below the predicted ordetermined T_(m) with washes of higher stringency, if desired.

The term ‘inhibit’ or ‘inhibiting’, in relationship to the term‘response’ means that a response is decreased or prevented in thepresence of a compound as opposed to in the absence of the compound. Theterm inhibit or inhibiting more generally refers to the relativereduction, decrease, or prevention of an activity or measurablephenomenon, particularly in the presence of a compound versus in theabsence of a compound.

The term ‘inhibition’ refers to the reduction, down regulation of aprocess or the elimination of a stimulus for a process, which results inthe absence or minimization of the expression or activity of a proteinor polypeptide.

The term ‘induction’ refers to the inducing, up-regulation, orstimulation of a process, which results in the expression or activity ofa protein or polypeptide.

The term ‘ligand’ means a molecule, including an endogenous, naturallyoccurring or synthetic, non-natural molecules, specific for anendogenous, naturally occurring receptor.

The term ‘pharmaceutically acceptable salts’ refers to the non-toxic,inorganic and organic acid addition salts, and base addition salts, ofcompounds which inhibit the expression or activity of TARGETS asdisclosed herein. These salts can be prepared in situ during the finalisolation and purification of compounds useful in the present invention.

The term ‘polypeptide’ relates to proteins (such as TARGETS),proteinaceous molecules, fragments of proteins, monomers, subunits orportions of polymeric proteins, peptides, oligopeptides and enzymes(such as kinases, proteases, GPCR's etc.).

The term ‘polynucleotide’ means a polynucleic acid, in single or doublestranded form, and in the sense or antisense orientation, complementarypolynucleic acids that hybridize to a particular polynucleic acid understringent conditions, and polynucleotides that are homologous in atleast about 60 percent of its base pairs, and more particularly 70percent of its base pairs are in common, most particularly 90 percent,and in a particular embodiment, 100 percent of its base pairs. Thepolynucleotides include polyribonucleic acids, polydeoxyribonucleicacids, and synthetic analogues thereof. It also includes nucleic acidswith modified backbones such as peptide nucleic acid (PNA),polysiloxane, and 2′-O-(2-methoxy)ethylphosphorothioate. Thepolynucleotides are described by sequences that vary in length, thatrange from about 10 to about 5000 bases, particularly about 100 to about4000 bases, more particularly about 250 to about 2500 bases. Onepolynucleotide embodiment comprises from about 10 to about 30 bases inlength. A particular embodiment of polynucleotide is thepolyribonucleotide of from about 17 to about 22 nucleotides, morecommonly described as small interfering RNAs (siRNAs—double strandedsiRNA molecules or self-complementary single-stranded siRNA molecules(shRNA)). Another particular embodiment are nucleic acids with modifiedbackbones such as peptide nucleic acid (PNA), polysiloxane, and2′-O-(2-methoxy)ethylphosphorothioate, or including non-naturallyoccurring nucleic acid residues, or one or more nucleic acidsubstituents, such as methyl-, thio-, sulphate, benzoyl-, phenyl-,amino-, propyl-, chloro-, and methanocarbanucleosides, or a reportermolecule to facilitate its detection. Polynucleotides herein areselected to be ‘substantially’ complementary to different strands of aparticular target DNA sequence. This means that the polynucleotides mustbe sufficiently complementary to hybridize with their respectivestrands. Therefore, the polynucleotide sequence need not reflect theexact sequence of the target sequence. For example, a non-complementarynucleotide fragment may be attached to the 5′ end of the polynucleotide,with the remainder of the polynucleotide sequence being complementary tothe strand. Alternatively, non-complementary bases or longer sequencescan be interspersed into the polynucleotide, provided that thepolynucleotide sequence has sufficient complementarity with the sequenceof the strand to hybridize therewith under stringent conditions or toform the template for the synthesis of an extension product.

The term ‘preventing’ or ‘prevention’ refers to a reduction in risk ofacquiring or developing a disease or disorder (i.e., causing at leastone of the clinical symptoms of the disease not to develop) in a subjectthat may be exposed to a disease-causing agent, or predisposed to thedisease in advance of disease onset.

The term ‘prophylaxis’ is related to and encompassed in the term‘prevention’, and refers to a measure or procedure the purpose of whichis to prevent, rather than to treat or cure a disease. Non-limitingexamples of prophylactic measures may include the administration ofvaccines; the administration of low molecular weight heparin to hospitalpatients at risk for thrombosis due, for example, to immobilization; andthe administration of an anti-malarial agent such as chloroquine, inadvance of a visit to a geographical region where malaria is endemic orthe risk of contracting malaria is high.

The term ‘solvate’ means a physical association of a compound useful inthis invention with one or more solvent molecules. This physicalassociation includes hydrogen bonding. In certain instances the solvatewill be capable of isolation, for example when one or more solventmolecules are incorporated in the crystal lattice of the crystallinesolid. “Solvate” encompasses both solution-phase and isolable solvates.Representative solvates include hydrates, ethanolates and methanolates.

The term ‘subject’ includes humans and other mammals.

‘Therapeutically effective amount’ means that amount of a drug,compound, expression inhibitory agent, or pharmaceutical agent that willelicit the biological or medical response of a subject that is beingsought by a medical doctor or other clinician.

The term ‘treating’ or ‘treatment’ of any disease or disorder refers, inone embodiment, to ameliorating the disease or disorder (i.e., arrestingthe disease or reducing the manifestation, extent or severity of atleast one of the clinical symptoms thereof). In another embodiment‘treating’ or ‘treatment’ refers to ameliorating at least one physicalparameter, which may not be discernible by the subject. In yet anotherembodiment, ‘treating’ or ‘treatment’ refers to modulating the diseaseor disorder, either physically, (e.g., stabilization of a discerniblesymptom), physiologically, (e.g., stabilization of a physicalparameter), or both. In a further embodiment, ‘treating’ or ‘treatment’relates to slowing the progression of the disease.

The term “vectors” also relates to plasmids as well as to viral vectors,such as recombinant viruses, or the nucleic acid encoding therecombinant virus.

The term “vertebrate cells” means cells derived from animals havingvertera structure, including fish, avian, reptilian, amphibian,marsupial, and mammalian species. Preferred cells are derived frommammalian species, and most preferred cells are human cells. Mammaliancells include feline, canine, bovine, equine, caprine, ovine, porcinemurine, such as mice and rats, and rabbits.

The term ‘TARGET’ or ‘TARGETS’ means the protein(s) identified inaccordance with the assays described herein and determined to beinvolved ECM and/or cartilage degradation. The term TARGET or TARGETSincludes and contemplates alternative species forms, isoforms, andvariants, such as splice variants, allelic variants, alternate in frameexons, and alternative or premature termination or start sites,including known or recognized isoforms or variants thereof such asindicated in Table 1.

The term ‘disease characterized by ECM and/or cartilage degradation’refers to a disease or condition which involves, results at least inpart from, or includes a breakdown of the extracellular matrix or abreakdown in cartilage or wherein the degradation of, degeneration of,or loss of cartilage and/or ECM exceeds the generation or regenerationof cartilage and/or ECM. The term includes, but is not limited to,exemplary diseases selected from osteoarthritis, rheumatoid arthritis,psoriatic arthritis, juvenile rheumatoid arthritis, gouty arthritis,septic or infectious arthritis, reactive arthritis, reflex sympatheticdystrophy, algodystrophy, Tietze syndrome or costal chondritis,fibromyalgia, osteochondritis, neurogenic or neuropathic arthritis,arthropathy, endemic forms of arthritis like osteoarthritis deformansendemica, Mseleni disease, and Handigodu disease; degeneration resultingfrom fibromyalgia, systemic lupus erythematosus, scleroderma, ankylosingspondylitis, congenital cartilage malformations, including hereditarychondrolysis, chondrodysplasias and pseudoachondrodysplasias, andcongenital cartilage malformation related diseases for example microtia,anotia, and metaphyseal chondrodysplasia.

The term ‘disease characterized by inflammation’ refers to a diseasewhich involves, results at least in part from or includes inflammation.The term includes, but is not limited to, exemplary diseases selectedfrom allergic airways disease (e.g. asthma, rhinitis), autoimmunediseases, transplant rejection, Crohn's disease, rheumatoid arthritis,psoriasis, juvenile idiopathic arthritis, colitis, and inflammatorybowel diseases.

TARGETS

The present invention is based on the present inventors' discovery thatTARGETS are factors in the regulation of catabolic processes ofchondrocytes, and in particular factors whose inhibition leads to adecrease in the catabolism of cartilage and/or ECM. Such a decrease maybe monitored by following the expression of proteins related tocartilage and/or ECM degradation, for example but without limitation,MMP1, MMP3, MMP8, MMP13, MMP14, and/or ADAMTS4, in particular MMP13. Theterm “TARGET” or “TARGETS” means the proteins identified in accordancewith the assay described below to be involved in the inhibition of ECMand/or cartilage degradation.

The TARGETS listed in Table 1 below were identified herein as involvedin the pathway that regulates or modulates the catabolism of cartilage,particularly as involved in inhibiting the catabolism of cartilage,therefore, inhibitors of these TARGETS are able to inhibit degradationof cartilage and are of use in the prevention and/or treatment ofdiseases characterized by cartilage degradation.

The TARGETS are also factors in inflammatory processes, in particularthey were identified as being involved in a reduction of the response topro-inflammatory stimuli, particularly the response to IL1 stimulation,therefore inhibitors of these TARGETS are able to inhibit inflammatoryprocesses and are of use in the prevention and/or treatment of diseasescharacterized by inflammation.

Therefore in one aspect, the present invention relates to a method forassaying for compounds that decrease ECM and/or cartilage degradation orinhibit inflammation, comprising contacting the compound with apolypeptide comprising an amino acid sequence of the polypeptides of SEQID NO: 22-42 (“TARGETS”) or a fragment thereof under conditions thatallow said polypeptide to bind to the compound, and detecting theformation of a complex between the polypeptide and the compound. Inparticular said method is used to identify an agent that inhibits thedegradation of cartilage. In particular said method may be used toidentify drug candidate compounds that inhibit the degradation ofcartilage via chondrocytes. In an alternative embodiment the method isused to identify an agent that inhibits inflammation. In an alternativeembodiment the method is used to identify an agent that inhibits ECMdegradation. One preferred means of measuring the complex formation isto determine the binding affinity of said compound to said polypeptide.

More particularly, the invention relates to a method for identifying anagent that reduces degradation of ECM and/or cartilage by chondrocytes,the method comprising:

-   -   (a) contacting a population of chondrocyte cells with one or        more compound(s) that exhibits binding affinity for a TARGET        polypeptide, or fragment thereof, and    -   (b) measuring a compound-polypeptide property related to the        inhibition of ECM and/or cartilage degradation.

In a further aspect of the present invention said method is used toidentify a compound that modulates the expression of or activity of oneor more cartilage degrading enzyme, including for example MMP1, MMP3,MMP8, MMP13, MMP14, and/or ADAMTS4. In particular the inhibition ofMMP13 expression may be measured.

In a further aspect of the present invention said method is used toidentify a compound that modulates the expression or activity of one ormore inflammatory cytokines, including for example IL-1b, IL-6, IL-8,IL-11, TNFα and/or LIF

In a further aspect, the present invention relates to a method forassaying for drug candidate compounds that inhibit cartilage and/or ECMdegradation said method comprising contacting the compound with apolypeptide comprising an amino acid sequence selected from SEQ ID NO:22-42, or a fragment thereof, under conditions that allow said compoundto modulate the activity or expression of the polypeptide, anddetermining the activity or expression of the polypeptide. In particularsaid method may be used to identify drug candidate compounds capable ofsuppressing expression of MMP1, MMP3, MMP8, MMP13, MMP14, and/orADAMTS4, in particular suppressing the expression of MMP13. Inparticular said method may be to identify drug candidate compoundscapable of suppressing the expression of MMP1, MMP3, MMP8, MMP13, MMP14,and/or ADAMTS4, in particular MMP13, in normal human articularchondrocytes (NHACs). One particular means of measuring the activity orexpression of the polypeptide is to determine the amount of saidpolypeptide using a polypeptide binding agent, such as an antibody, orto determine the activity of said polypeptide in a biological orbiochemical measure, for instance the amount of phosphorylation of atarget of a kinase polypeptide, or the amount of degradation by anenzyme or on an enzyme's substrate polypeptide.

The compound-polypeptide property referred to above is related to theinhibition of ECM and/or cartilage degradation, and is a measurablephenomenon chosen by the person of ordinary skill in the art. Themeasurable property may e.g. be the binding affinity for a peptidedomain of the polypeptide TARGET or the level of any one of a number ofbiochemical marker levels of decreased catabolism by chondrocytes.Catabolic inhibition of chondrocytes can e.g. be measured by measuringthe level of proteins and other molecules that are induced as part ofthe degradation pathway. In particular, the level of MMP13 may bemeasured.

In addition, compound-polypeptide properties related to the inhibitionof ECM and/or cartilage degradation may be measured in normal humanarticular chondrocytes (NHACs), SW1353 (chondrosarcoma cells),fibroblasts (e.g. synovial fibroblasts), or differentiating mesenchymalstem cell cultures. To some extent, such properties could be measured incells displaying expression of cartilage degrading enzymes, includingMMP1, MMP3, MMP8, MMP13, MMP14, and/or ADAMTS4 expression. However, suchproperties are also measured in alternative cell systems. For example,in situ binding assays that determine the affinity of compounds to bindto polypeptides of the invention are performed using any cell type thatexpresses the polypeptide. Expression of the polypeptide is exogenous orendogenous. Furthermore, when the compound-polypeptide property isactivation of a biological pathway, any cell that contains the pathwaycellular components is used to measure the compound-polypeptideproperty. The cell may inherently contain these components or may beengineered to express one or more component or express a variant of acomponent which is labeled or measurable. For example, induction ofMMP13 in response to IL-1 stimulation of NHACs is indicative ofcartilage and/or ECM degradation. Specifically, cells can be engineeredto contain a reporter molecule activated by the MMP13 promoters. In thisway alternative cells can be used to measure a property indicative ofinhibition of cartilage and/or ECM degradation.

In an additional aspect, the present invention relates to a method forassaying for drug candidate compounds that inhibit cartilage and/or ECMdegradation, said method, comprising contacting the compound with anucleic acid encoding a TARGET polypeptide, including a nucleic acidsequence selected from SEQ ID NO: 1-21, or a fragment thereof, underconditions that allow said nucleic acid to bind to or otherwiseassociate with the compound, and detecting the formation of a complexbetween the nucleic acid and the compound. In particular, said methodmay be used to identify drug candidate compounds able to reduce thelevel of proteins and other molecules that are induced as part of thedegradation pathway. In particular, said method may be used to identifydrug candidate compounds able to reduce the level of MMP1, MMP3, MMP8,MMP13, MMP14, and/or ADAMTS4. In particular, said method may be used toidentify drug candidate compounds able to reduce the level of MMP13. Oneparticular means of measuring the complex formation is to determine thebinding affinity of said compound to said nucleic acid or the presenceof a complex by virtue of resistance to nucleases or by gel mobilityassays. Alternatively, complex formation may be determined by inhibitionof nucleic acid transcription or translation.

The invention relates to a method for identifying a compound thatdecreases the degradation of cartilage and/or ECM, said methodcomprising the steps of: culturing a population of cells expressing apolypeptide of any one of those listed in Table 1, or a functionalfragment or derivative thereof; determining a first level of MMP1, MMP3,MMP8, MMP13, MMP14, and/or ADAMTS4 expression in response to IL-1stimulation in said population of cells; exposing said population ofcells to a compound, or a mixture of compounds; determining the level ofMMP1, MMP3, MMP8, MMP13, MMP14, and/or ADAMTS4 expression in response toIL-1 stimulation in said population of cells during or after exposure ofsaid population of cells to the compound, or the mixture of compounds;and identifying the compound that decreases the expression of MMP1,MMP3, MMP8, MMP13, MMP14, and/or ADAMTS4 is predictive of a decrease incartilage and/or ECM degradation. In a specific embodiment, thecartilage degrading enzyme measured in the method above is MMP13.

More particularly, the invention relates to a method for identifying anagent that reduces inflammation, the method comprising:

-   -   (a) contacting a population of cells with one or more        compound(s) that exhibits binding affinity for a TARGET        polypeptide, or fragment thereof, and    -   (b) measuring a compound-polypeptide property related to the        inhibition of inflammation.

In one embodiment the compound-polypeptide property related to theinhibition of inflammation is the response of the cells to IL1stimulation. In a further embodiment the agent is able to reduce theresponse of the cells to IL1 stimulation.

The invention also relates to a method for identifying a compound thatdecreases the expression and/or activity of any one of the polypeptideslisted in Table 1, said method comprising the steps of: culturing apopulation of cells expressing said polypeptide, or a fragment, or aderivative thereof; determining a first level of expression and/oractivity of said polypeptide; exposing said population of cells to acompound, or a mixture of compounds; determining the level of expressionand/or activity of said polypeptide during or after exposure of saidpopulation of cells to the compound, or the mixture of compounds; andidentifying the compound that decreases the expression and/or activityof said polypeptide. If the polypeptide activity is not readilymeasurable, the identification of the compound may benefit from an extrastep comprising exposing said population of cells to an agonist of saidpolypeptide. Furthermore, the methods of the present invention maycomprise the step of introducing a gene encoding any one of thepolypeptides listed in Table 1, in said population of cells. Forhigh-throughput purposes it may be beneficial to have the gene stablyintegrated in the genome of said cells.

In a preferred embodiment, the level of inhibition of cartilage and/orECM degradation is determined by measuring the expression level of amarker gene, wherein a particular marker gene encodes MMP1, MMP3, MMP8,MMP13, MMP14, and/or ADAMTS4. In a specific embodiment, the expressionand/or activity of MMP13 is measured. In a specific embodiment, theexpression and/or activity of MMP13 is decreased.

In a particular embodiment of the invention, the TARGET polypeptidecomprises an amino acid sequence selected from the group consisting ofSEQ ID No: 22-42 as listed in Table 1. In an embodiment of theinvention, the nucleic acid capable of encoding the TARGET polypeptidecomprises a nucleic acid sequence selected from the group consisting ofSEQ ID NO: 1-21 as listed in Table 1. Table 1 provides TARGET exemplaryhuman nucleic acid and protein sequence, including recognized variantsor isoforms where more than one accession number and SEQ ID NO: isindicated. Isoforms or variants of the TARGET(S) include nucleic acid orproteins with or utilizing alternate in frame exons, alternativesplicing or splice variants, and alternative or premature terminationvariants.

TABLE 1 TARGETS Target GenBank SEQ ID SEQ ID Gene Nucleic NO: GenBankNO: Symbol Acid Acc #: DNA Protein Acc # Protein: TARGET NAME Class METNM_000245 1 NP_000236 22 met proto-oncogene Kinase (hepatocyte growthfactor receptor) STK32B NM_018401 2 NP_060871 23 serine/threonine Kinasekinase 32B GPR34 NM_005300 3 NP_005291 24 G protein-coupled NM_0010335134 NP_001028685 25 receptor 34 GPCR NM_001033514 5 NP_001028686 26 GPR43NM_005306 6 NP_005297 27 G protein-coupled GPCR receptor 43 MAP2K2NM_030662 7 NP_109587 28 mitogen-activated Kinase protein kinase kinase2 ADAMTS6 NM_014273 8 NP_922932 29 a disintegrin-like Protease andmetalloprotease (reprolysin type) with thrombospondin type 1 motif, 6KCNN4 NM_002250 9 NP_002241 30 potassium Ion intermediate/small Channelconductance calcium-activated channel, subfamily N, member 4 ADAM15NM_003815 10 NP_003806 31 a disintegrin and NM_207191 11 NP_997074 32metalloproteinase NM_207194 12 NP_997077 33 domain 15 Protease NM_20719513 NP_997078 34 (metargidin) NM_207196 14 NP_997079 35 NM_207197 15NP_997080 36 MAP4K1 NM_007181 16 NP_009112 37 mitogen-activated Kinaseprotein kinase kinase kinase kinase 1 MC3R NM_019888 17 NP_063941 38melanocortin 3 GPCR receptor EPHA5 NM_004439 18 NP_004430 39 EPHreceptor A5 Kinase NM_182472 19 NP_872272 40 CSNK1G2 NM_001319 20NP_001310 41 casein kinase 1, Kinase gamma 2 EDG4 NM_004720 21 NP_00471142 endothelial GPCR differentiation, lysophosphatidic acid G-protein-coupled receptor, 4

The present invention provides in one particular embodiment methods foridentifying novel compounds, wherein the polypeptide is a GPCR. If so,the expression and/or activity of said GPCR is preferably determined bymeasuring the level of a second messenger. Preferred second messengersare cyclic AMP, Ca²⁺ or both. Typically, the level of the secondmessenger is determined with a reporter gene under the control of apromoter that is responsive to the second messenger, wherein it ispreferred that the promoter is a cyclic AMP-responsive promoter, anNF-KB responsive promoter, or a NF-AT responsive promoter, and whereinthe reporter gene is selected from the group consisting of: alkalinephosphatase, GFP, eGFP, dGFP, luciferase and β-galactosidase. ExemplaryTARGETs which are GPCRs are listed in Table 1 and include GPR34, GPR43,MC3R and EDG4.

In another particular embodiment, the invention provides methods foridentifying novel compounds, wherein the polypeptide is a kinase or aphosphatase. Preferably, the activity of said kinase or phosphatase isdetermined by measuring the level of phosphorylation of a substrate ofsaid kinase or phosphatase. Exemplary TARGETS which are kinases arelisted in Table 1 and include MET, STK32B, MAP2K2, MAP4K1, EPHA5, andCSNK1G2, Preferred TARGETS which are kinases are selected from the groupconsisting of STK32B, EPHA5 and CSNK1G2. Particularly preferred TARGETSwhich are kinases are EPHA5 and CSNK1G2.

In yet another particular embodiment, the invention provides methods foridentifying novel compounds, wherein the polypeptide is a protease.Preferably, the activity of said protease is measured by determining thelevel of cleavage of a substrate of said protease. Exemplary TARGETSwhich are proteases are listed in Table 1 and include ADAMTS6, andADAM15.

In yet another particular embodiment, the invention provides methods foridentifying novel compounds, wherein the polypeptide is an ion channel.An exemplary TARGET which is an ion channel is listed in Table 1, KCNN4.

Methods for determining second messenger levels, use of the reportergenes and second-messenger responsive promoters as well as phosphataseassays and protease assays are well known in the art and not furtherelaborated upon herein.

In a preferred embodiment, the compound that inhibits the polypeptideexhibits a binding affinity to the polypeptide of at most 10 micromolar.

In a preferred embodiment of the invention, the polypeptide TARGETcomprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 22-42 (Table 1).

Depending on the choice of the skilled artisan, the present assay methodmay be designed to function as a series of measurements, each of whichis designed to determine whether the drug candidate compound is indeedacting on the polypeptide to thereby inhibit the degradation ofcartilage and/or ECM or to inhibit inflammation. For example, an assaydesigned to determine the binding affinity of a compound to thepolypeptide, or fragment thereof, may be necessary, but not sufficient,to ascertain whether the test compound would be useful for decreasingECM and/or cartilage degradation when administered to a subject, oralternatively to ascertain whether the test compound would be useful fordecreasing inflammation. Nonetheless, such binding information would beuseful in identifying a set of test compounds for use in an assay thatwould measure a different property, further up the biochemical pathway,such as the MMP13 assay described below or an assay using cartilageexplants. Such second assay may be designed to confirm that the testcompound, having binding affinity for the polypeptide, actuallydecreases the degradation of cartilage and/or ECM or reducesinflammation, in vitro or in vivo.

Suitable controls should always be in place to insure against falsepositive readings. In a particular embodiment of the present inventionthe screening method comprises the additional step of comparing thecompound to a suitable control. In one embodiment, the control may be acell or a sample that has not been in contact with the test compound. Inan alternative embodiment, the control may be a cell that does notexpress the TARGET; for example in one aspect of such an embodiment thetest cell may naturally express the TARGET and the control cell may havebeen contacted with an agent, e.g. an siRNA, which inhibits or preventsexpression of the TARGET. Alternatively, in another aspect of such anembodiment, the cell in its native state does not express the TARGET andthe test cell has been engineered so as to express the TARGET, so thatin this embodiment, the control could be the untransformed native cell.The control may also or alternatively utilize a known mediator ofinflammation or ECM and/or cartilage degradation, such as cells treatedwith cytokines e.g. IL1, TNFα, OSM, or other inflammatory mediators(e.g. LPA or reactive oxygen species), prostaglandins, or leukotrienes.Whilst exemplary controls are described herein, this should not be takenas limiting; it is within the scope of a person of skill in the art toselect appropriate controls for the experimental conditions being used.

The order of taking these measurements is not believed to be critical tothe practice of the present invention, which may be practiced in anyorder. For example, one may first perform a screening assay of a set ofcompounds for which no information is known respecting the compounds'binding affinity for the polypeptide. Alternatively, one may screen aset of compounds identified as having binding affinity for a polypeptidedomain, or a class of compounds identified as being an inhibitor of thepolypeptide. However, for the present assay to be meaningful to theultimate use of the drug candidate compounds, a direct measurement ofinflammation, ECM degradation and/or cartilage degradation will bevaluable. Validation studies including controls, and measurements ofbinding affinity to the polypeptides of the invention are nonethelessuseful in identifying a compound useful in any therapeutic or diagnosticapplication.

Analogous approaches based on art-recognized methods and assays may beapplicable with respect to the TARGETS and compounds in any of variousdisease(s) characterized by cartilage and/or ECM degradation. An assayor assays may be designed to confirm that the test compound, havingbinding affinity for the TARGET, inhibits the degradation of cartilageand/or ECM. In one such method the expression and/or activity of acartilage degradative enzyme such as a collagenase is measured. In oneparticular such method the expression and/or activity of MMP1, MMP3,MMP8, MMP13, MMP14, and/or ADAMTS4 is measured. In one particular suchmethod the expression and/or activity of MMP13 is measured.

Analogous approaches based on art-recognized methods and assays may beapplicable with respect to the TARGETS and compounds in any of variousdisease(s) characterized by inflammation. An assay or assays may bedesigned to confirm that the test compound, having binding affinity forthe TARGET, inhibits inflammation. In one such method the expressionand/or activity of a cartilage degradative enzyme such as a collagenaseis measured. In one particular such method the expression and/oractivity of MMP1, MMP3, MMP8, MMP13, MMP14, and/or ADAMTS4 is measured.In one particular such method the expression and/or activity of MMP13 ismeasured. In an alternative method the level of one or more inflammatorycytokines selected from IL-1b, IL-6, IL-8, IL-11, TNFα and/or LIF aremeasured.

The present assay method may be practiced in vitro, using one or more ofthe TARGET proteins, or fragments thereof, including monomers, portionsor subunits of polymeric proteins, peptides, oligopeptides andenzymatically active portions thereof.

The binding affinity of the compound with the polypeptide TARGET can bemeasured by methods known in the art, such as using surface plasmonresonance biosensors (Biacore), by saturation binding analysis with alabeled compound (e.g. Scatchard and Lindmo analysis), by differentialUV spectrophotometer, fluorescence polarization assay, FluorometricImaging Plate Reader (FLIPR®) system, Fluorescence resonance energytransfer, and Bioluminescence resonance energy transfer. The bindingaffinity of compounds can also be expressed in dissociation constant(Kd) or as IC₅₀ or EC₅₀. The IC₅₀ represents the concentration of acompound that is required for 50% inhibition of binding of anotherligand to the polypeptide. The EC₅₀ represents the concentrationrequired for obtaining 50% of the maximum effect in any assay thatmeasures TARGET function. The dissociation constant, Kd, is a measure ofhow well a ligand binds to the polypeptide, it is equivalent to theligand concentration required to saturate exactly half of thebinding-sites on the polypeptide. Compounds with a high affinity bindinghave low Kd, IC₅₀ and EC₅₀ values, i.e. in the range of 100 nM to 1 pM;a moderate to low affinity binding relates to a high Kd, IC₅₀ and EC₅₀values, i.e. in the micromolar range.

The present assay method may also be practiced in a cellular assay, Ahost cell expressing TARGET can be a cell with endogenous expression ora cell over-expressing the TARGET e.g. by transduction. When theendogenous expression of the polypeptide is not sufficient to determinea baseline that can easily be measured, one may use using host cellsthat over-express TARGET. Over-expression has the advantage that thelevel of the TARGET substrate end products is higher than the activitylevel by endogenous expression. Accordingly, measuring such levels usingpresently available techniques is easier. In one such cellular assay,the biological activity of TARGET may be measured by following theproduction of cartilage component synthesis.

One embodiment of the present method for identifying a compound thatinhibits inflammation, ECM and/or cartilage degradation comprisesculturing a population of cells expressing a TARGET polypeptide, or afragment or derivative thereof; determining a first level of MMP1, MMP3,MMP8, MMP13, MMP14, and/or ADAMTS4, in particular MMP13, expressionand/or activity in said population of cells on activation of thepopulation of cells (e.g. by stimulation using IL1); exposing saidpopulation of cells to a compound, or a mixture of compounds;determining a second level of expression or activity of MMP1, MMP3,MMP8, MMP13, MMP14, and/or ADAMTS4, in particular MMP13, in saidpopulation of cells after the same activation, during or after exposureof said population of cells to said compound, or the mixture of saidcompounds; and identifying the compound(s) that suppress the expressionand/or activity of MMP1, MMP3, MMP8, MMP13, MMP14, and/or ADAMTS4, inparticular MMP13. In a specific embodiment, the cells are chondrocytes.In a specific embodiment the cells are mammalian cells. In a specificembodiment the cells are human cells.

The expression and/or activity of MMP1, MMP3, MMP8, MMP13, MMP14, and/orADAMTS4 can be determined by methods known in the art such as themethods as described herein.

The present inventors identified TARGET genes involved in the inhibitionof inflammation, cartilage and/or ECM degradation by using a‘knock-down’ library. This type of library is a screen in which siRNAmolecules are transduced into cells by recombinant adenoviruses, whichsiRNA molecules inhibit or repress the expression of a specific gene aswell as expression and activity of the corresponding gene product in acell. Each siRNA in a viral vector corresponds to a specific naturalgene. By identifying a siRNA that inhibits the degradation of cartilage,as measured by suppression of the expression of MMP13, a directcorrelation can be drawn between the specific gene expression and thepathway for inhibiting inflammation, cartilage and/or ECM degradation.The TARGET genes identified using the knock-down library (the proteinexpression products thereof herein referred to as “TARGET” polypeptides)are then used in the present inventive method for identifying compoundsthat can be used in the treatment of diseases associated with theinflammation or the degradation of ECM and/or cartilage. Indeed, shRNAcompounds comprising the sequences listed in Table 2 (SEQ ID NOs: 43-57)inhibit the expression and/or activity of these TARGET genes anddecrease the expression of MMP13, confirming the role of the TARGETS inthe pathway leading to the degradation of cartilage and/or ECM orinflammation.

TABLE 2 KD TARGET sequences useful in the practiceof the present expression-inhibitory agent invention Target Gene KD SEQHit ID Symbol KD Target Sequence ID No: H54-001 MET CATGGCTCTAGTTGTCGAC43 H54-016 STK32B TATCCTGCTGGATGAACAC 44 H54-023 GPR34GTAGGAGTGAAAGCACTTC 45 H54-024 GPR43 TACTTGAACACGACTGAGC 46 H54-025GPR43 CTGCTACGAGAACTTCACC 47 H54-044 MAP2K2 GATGCTCACAAACCACACC 48H54-058 ADAMTS6 CTTTCAGCCTATGGCAAGC 49 H54-094 KCNN4 ATGATCCTGTATGACCTGC50 H54-127 ADAM15 TCCAAGATCTCCACCTGCC 51 H54-305 ADAM15GTTGGAGCTGGACGGTGAC 52 H54-140 MAP4K1 GATCCAGGACACCAAAGGC 53 H54-165MC3R CATCTTCGACTCCATGATC 54 H54-257 EPHA5 AGATCAGTAGGTGAATGGC 55 H54-269CSNK1G2 ACCAGGCGTTGAACTCCAC 56 H54-340 EDG4 TCTGCTGGTCATAGCAGCC 57

The present invention further relates to a method for identifying acompound that reduces inflammation and/or the degradation of cartilageand/or ECM, comprising:

-   -   (a) contacting a compound with a polypeptide comprising an amino        acid sequence selected from the group consisting of SEQ ID NO:        22-42;    -   (b) determining the binding affinity of the compound to the        polypeptide;    -   (c) contacting a population of mammalian cells expressing said        polypeptide with the compound that exhibits a binding affinity        of at least 10 micromolar; and    -   (d) identifying a compound that reduces response to IL-1 or that        reduces the synthesis of enzymes that act to degrade ECM and/or        cartilage and/or related markers that indicate the presence of        said enzymes.

In one aspect, the assay method includes contacting cells expressingsaid polypeptide with the compound that exhibits a binding affinity inthe micromolar range. In an aspect, the binding affinity exhibited is atleast 10 micromolar. In an aspect, the binding affinity is at least 1micromolar. In an aspect, the binding affinity is at least 500nanomolar.

The assay method may be based on the particular expression or activityof the TARGET polypeptide, including but not limited to an enzymeactivity. Assays for the protease TARGETs ADAMTS6 (SEQ ID NO: 29) orADAM15 (SEQ ID NOs: 31, 32, 33, 34, 35, or 36) may be based on proteaseactivity or expression. Assays for the kinase TARGETs identified as MET(SEQ ID NO: 22), STK32B (SEQ ID NO: 23), MAP2K2 (SEQ ID NO: 28), MAP4K1(SEQ ID NO: 37), EPHA5 (SEQ ID NOs: 39 or 40) and CSNK1G2 (SEQ ID NO:41), may be based on kinase or phosphatase activity or expression,including but not limited to phosphorylation or dephosphorylation of atarget protein. Assays for the GPCR TARGETs identified as GPR34 (SEQ IDNOs: 24, 25 or 26), GPR43 (SEQ ID NO: 27) MC3R (SEQ ID NO: 39) and EDG4(SEQ ID NO: 42), may be based on GPCR activity or expression, includingdownstream mediators or activators. Assays for the ion channel TARGETidentified as KCNN4 (SEQ ID NO: 30) may use techniques well known tothose of skill in the art including classical patch clamping,high-throughput fluorescence based or tracer based assays which measurethe ability of a compound to open or close an ion channel therebychanging the concentration of fluorescent dyes or tracers across amembrane or within a cell. The measurable phenomenon, activity orproperty may be selected or chosen by the skilled artisan. The person ofordinary skill in the art may select from any of a number of assayformats, systems or design one using his knowledge and expertise in theart.

Table 1 lists the TARGETS identified using applicants' knock-downlibrary in the MMP13 assay described below, including the class ofpolypeptides identified. TARGETS have been identified in polypeptideclasses including kinase, phophatase, protease, GPCR, and ion channel,for instance. Specific methods to determine the activity of a kinase bymeasuring the phosphorylation of a substrate by the kinase, whichmeasurements are performed in the presence or absence of a compound, arewell known in the art.

Specific methods to determine the inhibition by a compound by measuringthe cleavage of the substrate by the polypeptide, which is a protease,are well known in the art. Classically, substrates are used in which afluorescent group is linked to a quencher through a peptide sequencethat is a substrate that can be cleaved by the target protease. Cleavageof the linker separates the fluorescent group and quencher, giving riseto an increase in fluorescence.

Ion channels are membrane protein complexes and their function is tofacilitate the diffusion of ions across biological membranes. Membranes,or phospholipid bilayers, build a hydrophobic, low dielectric barrier tohydrophilic and charged molecules. Ion channels provide a highconducting, hydrophilic pathway across the hydrophobic interior of themembrane. The activity of an ion channel can be measured using classicalpatch clamping. High-throughput fluorescence-based or tracer-basedassays are also widely available to measure ion channel activity. Thesefluorescent-based assays screen compounds on the basis of their abilityto either open or close an ion channel thereby changing theconcentration of specific fluorescent dyes across a membrane. In thecase of the tracer based assay, the changes in concentration of thetracer within and outside the cell are measured by radioactivitymeasurement or gas absorption spectrometry.

G-protein coupled receptors (GPCR) are capable of activating an effectorprotein, resulting in changes in second messenger levels in the cell.The activity of a GPCR can be determined by measuring the activity levelof such second messengers. Two exemplary important and useful secondmessengers in the cell are cyclic AMP (cAMP) and Ca²⁺. The secondmessenger activity levels can be measured by methods known to personsskilled in the art, either directly by ELISA or radioactive technologiesor by using substrates that generate a fluorescent or luminescent signalwhen contacted with Ca²⁺ or indirectly by reporter gene analysis. Theactivity level of the one or more secondary messengers may typically bedetermined with a reporter gene controlled by a promoter, wherein thepromoter is responsive to the second messenger. Promoters known and usedin the art for such purposes are the cyclic-AMP responsive promoter thatis responsive for the cyclic-AMP levels in the cell, and the NF-ATresponsive promoter that is sensitive to cytoplasmic Ca²⁺-levels in thecell. The reporter gene typically has a gene product that is easilydetectable. The reporter gene can either be stably infected ortransiently transfected in the host cell. Useful reporter genes arealkaline phosphatase, enhanced green fluorescent protein, destabilizedgreen fluorescent protein, luciferase and β-galactosidase.

It should be understood that the cells expressing the polypeptides maybe cells which naturally express the polypeptides, or the cells may betransfected to express the polypeptides, as described above. Also, thecells may be transduced to overexpress the polypeptide, or may betransfected to express a non-endogenous form of the polypeptide, whichcan be differentially assayed or assessed.

In one particular embodiment the methods of the present inventionfurther comprise the step of contacting the population of cells with anagonist of the polypeptide. This is useful in methods wherein theexpression of the polypeptide in a certain chosen population of cells istoo low for a proper detection of its activity. By using an agonist thepolypeptide may be triggered, enabling a proper read-out if the compoundinhibits the polypeptide. Similar considerations apply to themeasurement of the release of inflammatory mediators. In a particularembodiment, the cells used in the present method are mammalian NHACs.The NHACs, in the assay contemplated, may be activated (e.g. bystimulation with IL1).

A method for identifying a compound that inhibits inflammation orcartilage and/or ECM degradation, comprising:

-   -   (a) contacting a compound with a polypeptide comprising an amino        acid sequence selected from the group consisting of SEQ ID NO:        22-42, and fragments thereof; and    -   (b) measuring a compound-polypeptide property related to        cartilage degradation.

In one embodiment of the present invention the method relates toidentifying a compound that inhibits the catabolic processes ofchondrocytes.

In one embodiment of the present invention the compound-polypeptideproperty related to inflammation or cartilage and/or ECM degradation isbinding affinity.

In one embodiment the compound-polypeptide property related toinflammation or cartilage and/or ECM degradation is the inhibition ofMMP1, MMP3, MMP8, MMP13, MMP14, and/or ADAMTS4 expression and/oractivity.

In one embodiment the compound-polypeptide property related toinflammation or cartilage and/or ECM degradation is the inhibition ofMMP13 expression and/or activity.

In one embodiment the compound-polypeptide property related toinflammation or cartilage and/or ECM degradation is the expression ofinflammatory cytokines such as IL-1b, IL-6, IL-8, IL-11, TNFα and/orLIF.

In one embodiment of the present invention the compound-polypeptideproperty related to inflammation or cartilage and/or ECM degradation isthe activity of said polypeptide. In particular, in one embodiment thecompound inhibits the activity of said polypeptide.

In one embodiment of the present invention the compound-polypeptideproperty related to inflammation or cartilage and/or ECM degradation isthe expression of said polypeptide. In particular, in one embodiment thecompound inhibits the expression of said polypeptide.

The present invention further relates to a method for identifying acompound that inhibits inflammation or cartilage and/or ECM degradation,wherein said compound exhibits at least a moderate binding affinity toan amino acid selected from the group of SEQ ID NOS: 22-42, said methodcomprising:

-   -   a) contacting a compound with a population of mammalian cells        expressing a polypeptide comprising an amino acid sequence        selected from the group consisting of SEQ ID NOS: 22-42, wherein        the cells have been activated;    -   b) determining the expression of MMP13 from said cells; and    -   c) identifying the compound that inhibits inflammation or        cartilage and/or ECM degradation as the compound which        suppresses the release of MMP13 from the cells.

In one such method the cells are activated by being contacted withpro-inflammatory factors. In a specific embodiment of the method thepro-inflammatory factors are selected from TNF-alpha, IL-1, OSM(oncostatin M), IL6, endothelin, bradykinin, LPA, leukotrienes,prostaglandins, LPS (lipo poly saccharides) or other TLR ligands, orcombinations thereof.

In one such method, the compound exhibits a binding affinity to an aminoacid selected from the group of SEQ ID NOS: 22-42 of at least 10micromolar.

The present invention further relates to a method for identifying acompound that inhibits inflammation or cartilage and/or ECM degradation,said method comprising:

-   -   a) contacting a compound with a polypeptide comprising an amino        acid sequence selected from the group consisting of SEQ ID NO:        22-42;    -   b) determining the binding affinity of the compound to the        polypeptide;    -   c) contacting a population of mammalian cells expressing said        polypeptide with the compound that exhibits a binding affinity        of at least 10 micromolar; and    -   d) identifying the compound that inhibits inflammation or        cartilage and/or ECM degradation.

The present invention further relates to a method for identifying acompound that inhibits inflammation or cartilage and/or ECM degradationsaid method comprising:

-   -   a) contacting a compound with a polypeptide comprising an amino        acid sequence selected from the group consisting of SEQ ID NO:        22-42;    -   b) determining the ability of the compound to inhibit the        expression or activity of the polypeptide;    -   c) contacting a population of mammalian cells expressing said        polypeptide with the compound that significantly inhibits the        expression or activity of the polypeptide; and    -   d) identifying the compound that inhibits inflammation or        cartilage and/or ECM degradation.

In a particular aspect of the present invention the methods describedabove include the additional step of comparing the compound to be testedto a control, where the control is a population of cells that have notbeen contacted with the test compound.

In a particular aspect of the present invention the methods describedabove include the additional step of comparing the compound to be testedto a control, where the control is a population of cells that do notexpress said polypeptide.

For high-throughput purposes, libraries of compounds may be used such asantibody fragment libraries, peptide phage display libraries, peptidelibraries (e.g. LOPAP™, Sigma Aldrich), lipid libraries (BioMol),synthetic compound libraries (e.g. LOPAC™, Sigma Aldrich, BioFocus DPI)or natural compound libraries (Specs, TimTec, BioFocus DPI).

Preferred drug candidate compounds are low molecular weight compounds.Low molecular weight compounds, i.e. with a molecular weight of 500Dalton or less, are likely to have good absorption and permeation inbiological systems and are consequently more likely to be successfuldrug candidates than compounds with a molecular weight above 500 Dalton(Lipinski et al., (2001)). Peptides comprise another preferred class ofdrug candidate compounds. Peptides may be excellent drug candidates andthere are multiple examples of commercially valuable peptides such asfertility hormones and platelet aggregation inhibitors. Natural productsare another preferred class of drug candidate compound. Such compoundsare found in and extracted from natural sources, and which maythereafter be synthesized. The lipids are another preferred class ofdrug candidate compound.

Another preferred class of drug candidate compounds is an antibody. Thepresent invention also provides antibodies directed against a TARGET.These antibodies may be endogenously produced to bind to the TARGETwithin the cell, or added to the tissue to bind to TARGET polypeptidepresent outside the cell. These antibodies may be monoclonal antibodiesor polyclonal antibodies. The present invention includes chimeric,single chain, domain antibodies, camelid antibodies, and humanizedantibodies, as well as FAb fragments and the products of a FAbexpression library, and Fv fragments and the products of an Fvexpression library. The antibodies may be neutralizing antibodies orantibodies that inhibit the activity of the TARGET or that block orinhibit binding of a ligand to the TARGET or of the TARGET to anotherprotein.

In certain embodiments, polyclonal antibodies may be used in thepractice of the invention. The skilled artisan knows methods ofpreparing polyclonal antibodies. Polyclonal antibodies can be raised ina mammal, for example, by one or more injections of an immunizing agentand, if desired, an adjuvant. Typically, the immunizing agent and/oradjuvant will be injected in the mammal by multiple subcutaneous orintraperitoneal injections. Antibodies may also be generated against theintact TARGET protein or polypeptide, or against a fragment, derivativesincluding conjugates, or other epitope of the TARGET protein orpolypeptide, such as the TARGET embedded in a cellular membrane, or alibrary of antibody variable regions, such as a phage display library.

It may be useful to conjugate the immunizing agent to a protein known tobe immunogenic in the mammal being immunized. Examples of suchimmunogenic proteins include but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. Examples of adjuvants that may be employed include Freund'scomplete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A,synthetic trehalose dicorynomycolate). One skilled in the art withoutundue experimentation may select the immunization protocol.

In some embodiments, the antibodies may be monoclonal antibodies.Monoclonal antibodies may be prepared using methods known in the art.The monoclonal antibodies of the present invention may be “humanized” toprevent the host from mounting an immune response to the antibodies. A“humanized antibody” is one in which the complementarity determiningregions (CDRs) and/or other portions of the light and/or heavy variabledomain framework are derived from a non-human immunoglobulin, but theremaining portions of the molecule are derived from one or more humanimmunoglobulins. Humanized antibodies also include antibodiescharacterized by a humanized heavy chain associated with a donor oracceptor unmodified light chain or a chimeric light chain, or viceversa. The humanization of antibodies may be accomplished by methodsknown in the art (see, e.g. Mark and Padlan, 1994). Transgenic animalsmay be used to express humanized antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries (Hoogenboom and Winter, 1991;Marks et al., 1991). The techniques of Cole, et al. and Boerner, et al.are also available for the preparation of human monoclonal antibodies(Cole, et al., (1985); Boerner, et al., 1991) Techniques known in theart for the production of single chain antibodies can be adapted toproduce single chain antibodies to the TARGET polypeptides and proteinsof the present invention. The antibodies may be monovalent antibodies.Methods for preparing monovalent antibodies are well known in the art.For example, one method involves recombinant expression ofimmunoglobulin light chain and modified heavy chain. The heavy chain istruncated generally at any point in the Fc region so as to prevent heavychain cross-linking. Alternatively; the relevant cysteine residues aresubstituted with another amino acid residue or are deleted so as toprevent cross-linking.

Bispecific antibodies are monoclonal, preferably human or humanized,antibodies that have binding specificities for at least two differentantigens and preferably for a cell-surface protein or receptor orreceptor subunit. In the present case, one of the binding specificitiesis for one domain of the TARGET; the other one is for another domain ofthe same or different TARGET.

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milsteinand Cuello, (1983)). Because of the random assortment of immunoglobulinheavy and light chains, these hybridomas (quadromas) produce a potentialmixture of ten different antibody molecules, of which only one has thecorrect bispecific structure. Affinity chromatography steps usuallyaccomplish the purification of the correct molecule. Similar proceduresare disclosed in Trauneeker et al., 1991.

According to another preferred embodiment, the assay method uses a drugcandidate compound identified as having a binding affinity for a TARGET,and/or has already been identified as having down-regulating activitysuch as antagonist activity vis-à-vis one or more TARGET.

In vivo animal models of arthritis or osteoarthritis or of inflammationor inflammatory diseases may be utilized by the skilled artisan tofurther or additionally screen, assess, and/or verify the agents orcompounds identified in the present invention, including furtherassessing TARGET modulation in vivo. Such animal models include, but arenot limited to, ulcerative colitis models, multiple sclerosis models(including EAE, lysolecithin-induced), arthritis models, allergic asthmamodels, airway inflammation models, and acute inflammation models.Osteoarthritis models include for example experimental osteoarthritisinduced in rabbits after sectioning of the knee anterior cruciateligament and in rats after tear of the medial collateral ligament.

The present invention further relates to a method for inducing anabolicstimulation of chondrocytes or reducing chondrocyte degradationcomprising contacting said cells with an expression inhibitory agentcomprising a polynucleotide sequence that complements at least about 17nucleotides of the polyribonucleotide comprising a nucleotide sequenceselected from the group consisting of SEQ ID NO: 1-21. In a preferredembodiment the expression-inhibitory agent comprises a polynucleotidesequence that complements a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 43-57.

Another aspect of the present invention relates to a method forinhibiting inflammation or cartilage and/or ECM degradation, comprisingcontacting said cell with an expression-inhibiting agent that inhibitsthe translation in the cell of a polyribonucleotide encoding a TARGETpolypeptide. A particular embodiment relates to a composition comprisinga polynucleotide including at least one antisense strand that functionsto pair the agent with the TARGET mRNA, and thereby down-regulate orblock the expression of TARGET polypeptide. The inhibitory agentpreferably comprises antisense polynucleotide, a ribozyme, or a smallinterfering RNA (siRNA), wherein said agent comprises a nucleic acidsequence complementary to, or engineered from, a naturally-occurringpolynucleotide sequence encoding a portion of a polypeptide comprisingan amino acid sequence selected from the group consisting of SEQ ID NO:22-42. In a preferred embodiment the expression-inhibiting agent iscomplementary to a polynucleotide sequence selected from the groupconsisting of SEQ ID NO: 1-21. In an especially preferred embodiment theexpression-inhibiting agent is complementary to a polynucleotidesequence selected from the group consisting of SEQ ID NO: 43-57.

An embodiment of the present invention relates to a method wherein theexpression-inhibiting agent is selected from the group consisting ofantisense RNA, antisense oligodeoxynucleotide (ODN), a ribozyme thatcleaves the polyribonucleotide coding for SEQ ID NO: 22-42, a smallinterfering RNA (siRNA, preferably shRNA) that is sufficientlycomplementary to a portion of the polyribonucleotide coding for SEQ IDNO: 22-42, such that the siRNA, preferably shRNA, interferes with thetranslation of the TARGET polyribonucleotide to the TARGET polypeptide.Preferably the expression-inhibiting agent is an antisense RNA,ribozyme, antisense oligodeoxynucleotide, or siRNA, preferably shRNA,complementary to a nucleotide sequence selected from the groupconsisting of SEQ ID NO: 1-21. In an especially preferred embodiment theexpression-inhibiting agent is complementary to a polynucleotidesequence selected from the group consisting of SEQ ID NO: 43-57.

A special embodiment of the present invention relates to a methodwherein the expression-inhibiting agent is a nucleic acid expressing theantisense RNA, antisense oligodeoxynucleotide (ODN), a ribozyme thatcleaves the polyribonucleotide coding for SEQ ID NO: 22-42, a smallinterfering RNA (siRNA, preferably shRNA) that is sufficientlycomplementary to a portion of the polyribonucleotide coding for SEQ IDNO: 22-42, such that the siRNA, preferably shRNA, interferes with thetranslation of the TARGET polyribonucleotide to the TARGET polypeptide.Preferably the nucleotide sequence is complementary to a polynucleotideselected from the group consisting of SEQ ID NO: 1-21. In an especiallypreferred embodiment nucleotide sequence is complementary to apolynucleotide selected from the group consisting of SEQ ID NO: 43-57.

The down regulation of gene expression using antisense nucleic acids canbe achieved at the translational or transcriptional level. Antisensenucleic acids of the invention are preferably nucleic acid fragmentscapable of specifically hybridizing with all or part of a nucleic acidencoding a TARGET polypeptide or the corresponding messenger RNA. Inaddition, antisense nucleic acids may be designed which decreaseexpression of the nucleic acid sequence capable of encoding a TARGETpolypeptide by inhibiting splicing of its primary transcript. Any lengthof antisense sequence is suitable for practice of the invention so longas it is capable of down-regulating or blocking expression of a nucleicacid coding for a TARGET. Preferably, the antisense sequence is at leastabout 17 nucleotides in length. The preparation and use of antisensenucleic acids, DNA encoding antisense RNAs and the use of oligo andgenetic antisense is known in the art.

One embodiment of expression-inhibitory agent is a nucleic acid that isantisense to a nucleic acid comprising SEQ ID NO: 1-21. For example, anantisense nucleic acid (e.g. DNA) may be introduced into cells in vitro,or administered to a subject in vivo, as gene therapy to inhibitcellular expression of nucleic acids comprising SEQ ID NO: 1-21.Antisense oligonucleotides preferably comprise a sequence containingfrom about 17 to about 100 nucleotides and more preferably the antisenseoligonucleotides comprise from about 18 to about 30 nucleotides.Antisense nucleic acids may be prepared from about 10 to about 30contiguous nucleotides complementary to a nucleic acid sequence selectedfrom the sequences of SEQ ID NO: 1-21.

The antisense nucleic acids are preferably oligonucleotides and mayconsist entirely of deoxyribo-nucleotides, modifieddeoxyribonucleotides, or some combination of both. The antisense nucleicacids can be synthetic oligonucleotides. The oligonucleotides may bechemically modified, if desired, to improve stability and/orselectivity. Since oligonucleotides are susceptible to degradation byintracellular nucleases, the modifications can include, for example, theuse of a sulfur group to replace the free oxygen of the phosphodiesterbond. This modification is called a phosphorothioate linkage.Phosphorothioate antisense oligonucleotides are water soluble,polyanionic, and resistant to endogenous nucleases. In addition, when aphosphorothioate antisense oligonucleotide hybridizes to its TARGETsite, the RN202-315NA duplex activates the endogenous enzymeribonuclease (RNase) H, which cleaves the mRNA component of the hybridmolecule.

In addition, antisense oligonucleotides with phosphoramidite andpolyamide (peptide) linkages can be synthesized. These molecules shouldbe very resistant to nuclease degradation. Furthermore, chemical groupscan be added to the 2′ carbon of the sugar moiety and the 5 carbon (C-5)of pyrimidines to enhance stability and facilitate the binding of theantisense oligonucleotide to its TARGET site. Modifications may include2′-deoxy, O-pentoxy, O-propoxy, O-methoxy, fluoro, methoxyethoxyphosphorothioates, modified bases, as well as other modifications knownto those of skill in the art.

Another type of expression-inhibitory agent that reduces the levels ofTARGETS is the ribozyme. Ribozymes are catalytic RNA molecules (RNAenzymes) that have separate catalytic and substrate binding domains. Thesubstrate binding sequence combines by nucleotide complementarity and,possibly, non-hydrogen bond interactions with its TARGET sequence. Thecatalytic portion cleaves the TARGET RNA at a specific site. Thesubstrate domain of a ribozyme can be engineered to direct it to aspecified mRNA sequence. The ribozyme recognizes and then binds a TARGETmRNA through complementary base pairing. Once it is bound to the correctTARGET site, the ribozyme acts enzymatically to cut the TARGET mRNA.Cleavage of the mRNA by a ribozyme destroys its ability to directsynthesis of the corresponding polypeptide. Once the ribozyme hascleaved its TARGET sequence, it is released and can repeatedly bind andcleave at other mRNAs.

Ribozyme forms include a hammerhead motif, a hairpin motif, a hepatitisdelta virus, group I intron or RNaseP RNA (in association with an RNAguide sequence) motif or Neurospora VS RNA motif. Ribozymes possessing ahammerhead or hairpin structure are readily prepared since thesecatalytic RNA molecules can be expressed within cells from eukaryoticpromoters (Chen, et al., 1992). A ribozyme of the present invention canbe expressed in eukaryotic cells from the appropriate DNA vector. Ifdesired, the activity of the ribozyme may be augmented by its releasefrom the primary transcript by a second ribozyme (Ventura, et al.,1993).

Ribozymes may be chemically synthesized by combining anoligodeoxyribonucleotide with a ribozyme catalytic domain (20nucleotides) flanked by sequences that hybridize to the TARGET mRNAafter transcription. The oligodeoxyribonucleotide is amplified by usingthe substrate binding sequences as primers. The amplification product iscloned into a eukaryotic expression vector.

Ribozymes are expressed from transcription units inserted into DNA, RNA,or viral vectors. Transcription of the ribozyme sequences are drivenfrom a promoter for eukaryotic RNA polymerase I (pol (I), RNA polymeraseII (pol II), or RNA polymerase III (pol III). Transcripts from pol II orpol III promoters will be expressed at high levels in all cells; thelevels of a given pol II promoter in a given cell type will depend onnearby gene regulatory sequences. Prokaryotic RNA polymerase promotersare also used, providing that the prokaryotic RNA polymerase enzyme isexpressed in the appropriate cells (Gao and Huang, 1993). It has beendemonstrated that ribozymes expressed from these promoters can functionin mammalian cells (Kashani-Sabet, et al., 1992).

A particularly preferred inhibitory agent is a small interfering RNA(siRNA, preferably shRNA). siRNA, preferably shRNA, mediates thepost-transcriptional process of gene silencing by double stranded RNA(dsRNA) that is homologous in sequence to the silenced RNA. siRNAaccording to the present invention comprises a sense strand of 17-25nucleotides complementary or homologous to a contiguous 17-25 nucleotidesequence selected from the group of sequences described in SEQ ID NO:1-21, preferably from the group of sequences described in SEQ ID No:43-57, and an antisense strand of 17-23 nucleotides complementary to thesense strand. Exemplary sequences are described as sequencescomplementary to SEQ ID NO: 43-57. The most preferred siRNA comprisessense and anti-sense strands that are 100 percent complementary to eachother and the TARGET polynucleotide sequence. Preferably the siRNAfurther comprises a loop region linking the sense and the antisensestrand.

A self-complementing single stranded siRNA molecule polynucleotideaccording to the present invention comprises a sense portion and anantisense portion connected by a loop region linker. Preferably, theloop region sequence is 4-30 nucleotides long, more preferably 5-15nucleotides long and most preferably 8 or 12 nucleotides long. In aspecific embodiment the linker sequence is UUGCUAUA. In an alternativespecific embodiment the linker sequence is GUUUGCUAUAAC (SEQ ID NO: 58).Self-complementary single stranded siRNAs form hairpin loops and aremore stable than ordinary dsRNA. In addition, they are more easilyproduced from vectors.

Analogous to antisense RNA, the siRNA can be modified to confirmresistance to nucleolytic degradation, or to enhance activity, or toenhance cellular distribution, or to enhance cellular uptake, suchmodifications may consist of modified internucleoside linkages, modifiednucleic acid bases, modified sugars and/or chemical linkage the siRNA toone or more moieties or conjugates. The nucleotide sequences areselected according to siRNA designing rules that give an improvedreduction of the TARGET sequences compared to nucleotide sequences thatdo not comply with these siRNA designing rules (For a discussion ofthese rules and examples of the preparation of siRNA, WO 2004/094636,and US 2003/0198627, are hereby incorporated by reference).

The present invention also relates to compositions, and methods usingsaid compositions, comprising a DNA expression vector capable ofexpressing a polynucleotide capable of inhibiting the degradation ofcartilage and described hereinabove as an expression inhibition agent.

A special aspect of these compositions and methods relates to thedown-regulation or blocking of the expression of a TARGET polypeptide bythe induced expression of a polynucleotide encoding an intracellularbinding protein that is capable of selectively interacting with theTARGET polypeptide. An intracellular binding protein includes anyprotein capable of selectively interacting, or binding, with thepolypeptide in the cell in which it is expressed and neutralizing thefunction of the polypeptide. Preferably, the intracellular bindingprotein is a neutralizing antibody or a fragment of a neutralizingantibody having binding affinity to an epitope of the TARGET polypeptideof SEQ ID NO: 22-42. More preferably, the intracellular binding proteinis a single chain antibody.

A special embodiment of this composition comprises theexpression-inhibiting agent selected from the group consisting ofantisense RNA, antisense oligodeoxynucleotide (ODN), a ribozyme thatcleaves the polyribonucleotide coding for SEQ ID NO: 22-42, and a smallinterfering RNA (siRNA) that is sufficiently homologous to a portion ofthe polyribonucleotide coding for SEQ ID NO: 22-42, such that the siRNAinterferes with the translation of the TARGET polyribonucleotide to theTARGET polypeptide.

The polynucleotide expressing the expression-inhibiting agent ispreferably included within a vector. The polynucleic acid is operablylinked to signals enabling expression of the nucleic acid sequence andis introduced into a cell utilizing, preferably, recombinant vectorconstructs, which will express the antisense nucleic acid once thevector is introduced into the cell. A variety of viral-based systems areavailable, including adenoviral, retroviral, adeno-associated viral,lentiviral, herpes simplex viral or a sendaviral vector systems, and allmay be used to introduce and express polynucleotide sequence for theexpression-inhibiting agents in TARGET cells.

Preferably, the viral vectors used in the methods of the presentinvention are replication defective. Such replication defective vectorswill usually lack at least one region that is necessary for thereplication of the virus in the infected cell. These regions can eitherbe eliminated (in whole or in part), or be rendered non-functional byany technique known to a person skilled in the art. These techniquesinclude the total removal, substitution, partial deletion or addition ofone or more bases to an essential (for replication) region. Suchtechniques may be performed in vitro (on the isolated DNA) or in situ,using the techniques of genetic manipulation or by treatment withmutagenic agents. Preferably, the replication defective virus retainsthe sequences of its genome, which are necessary for encapsidating, theviral particles.

In a preferred embodiment, the viral element is derived from anadenovirus. Preferably, the vehicle includes an adenoviral vectorpackaged into an adenoviral capsid, or a functional part, derivative,and/or analogue thereof. Adenovirus biology is also comparatively wellknown on the molecular level. Many tools for adenoviral vectors havebeen and continue to be developed, thus making an adenoviral capsid apreferred vehicle for incorporating in a library of the invention. Anadenovirus is capable of infecting a wide variety of cells. However,different adenoviral serotypes have different preferences for cells. Tocombine and widen the TARGET cell population that an adenoviral capsidof the invention can enter in a preferred embodiment, the vehicleincludes adenoviral fiber proteins from at least two adenoviruses.Preferred adenoviral fiber protein sequences are serotype 5, 17, 45 and51. Techniques or construction and expression of these chimeric vectorsare disclosed in US 2003/0180258 and US 2004/0071660, herebyincorporated by reference.

In a preferred embodiment, the nucleic acid derived from an adenovirusincludes the nucleic acid encoding an adenoviral late protein or afunctional part, derivative, and/or analogue thereof. An adenoviral lateprotein, for instance an adenoviral fiber protein, may be favorably usedto TARGET the vehicle to a certain cell or to induce enhanced deliveryof the vehicle to the cell. Preferably, the nucleic acid derived from anadenovirus encodes for essentially all adenoviral late proteins,enabling the formation of entire adenoviral capsids or functional parts,analogues, and/or derivatives thereof. Preferably, the nucleic acidderived from an adenovirus includes the nucleic acid encoding adenovirusE2A or a functional part, derivative, and/or analogue thereof.Preferably, the nucleic acid derived from an adenovirus includes thenucleic acid encoding at least one E4-region protein or a functionalpart, derivative, and/or analogue thereof, which facilitates, at leastin part, replication of an adenoviral derived nucleic acid in a cell.The adenoviral vectors used in the examples of this application areexemplary of the vectors useful in the present method of treatmentinvention.

Certain embodiments of the present invention use retroviral vectorsystems. Retroviruses are integrating viruses that infect dividingcells, and their construction is known in the art. Retroviral vectorscan be constructed from different types of retrovirus, such as, MoMuLV(“murine Moloney leukemia virus” MSV (“murine Moloney sarcoma virus”),HaSV (“Harvey sarcoma virus”); SNV (“spleen necrosis virus”); RSV (“Roussarcoma virus”) and Friend virus. Lentiviral vector systems may also beused in the practice of the present invention.

In other embodiments of the present invention, adeno-associated viruses(“AAV”) are utilized. The AAV viruses are DNA viruses of relativelysmall size that integrate, in a stable and site-specific manner, intothe genome of the infected cells. They are able to infect a widespectrum of cells without inducing any effects on cellular growth,morphology or differentiation, and they do not appear to be involved inhuman pathologies.

In the vector construction, the polynucleotide agents of the presentinvention may be linked to one or more regulatory regions. Selection ofthe appropriate regulatory region or regions is a routine matter, withinthe level of ordinary skill in the art. Regulatory regions includepromoters, and may include enhancers, suppressors, etc.

Promoters that may be used in the expression vectors of the presentinvention include both constitutive promoters and regulated (inducible)promoters. The promoters may be prokaryotic or eukaryotic depending onthe host. Among the prokaryotic (including bacteriophage) promotersuseful for practice of this invention are lac, lacZ, T3, T7, lambdaP_(r), P_(l), and trp promoters. Among the eukaryotic (including viral)promoters useful for practice of this invention are ubiquitous promoters(e.g. HPRT, vimentin, actin, tubulin), intermediate filament promoters(e.g. desmin, neurofilaments, keratin, GFAP), therapeutic gene promoters(e.g. MDR type, CFTR, factor VIII), tissue-specific promoters (e.g.actin promoter in smooth muscle cells, or Flt and Flk promoters activein endothelial cells), including animal transcriptional control regions,which exhibit tissue specificity and have been utilized in transgenicanimals: elastase I gene control region which is active in pancreaticacinar cells (Swift, et al. (1984) Cell 38:639-46; Ornitz, et al. (1986)Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, (1987)Hepatology 7:425-515); insulin gene control region which is active inpancreatic beta cells (Hanahan, (1985) Nature 315:115-22),immunoglobulin gene control region which is active in lymphoid cells(Grosschedl, et al. (1984) Cell 38:647-58; Adames, et al. (1985) Nature318:533-8; Alexander, et al. (1987) Mol. Cell. Biol. 7:1436-44), mousemammary tumor virus control region which is active in testicular,breast, lymphoid and mast cells (Leder, et al. (1986) Cell 45:485-95),albumin gene control region which is active in liver (Pinkert, et al.(1987) Genes and Devel. 1:268-76), alpha-fetoprotein gene control regionwhich is active in liver (Krumlauf, et al. (1985) Mol. Cell. Biol.,5:1639-48; Hammer, et al. (1987) Science 235:53-8), alpha 1-antitrypsingene control region which is active in the liver (Kelsey, et al. (1987)Genes and Devel., 1: 161-71), beta-globin gene control region which isactive in myeloid cells (Mogram, et al. (1985) Nature 315:338-40;Kollias, et al. (1986) Cell 46:89-94), myelin basic protein gene controlregion which is active in oligodendrocyte cells in the brain (Readhead,et al. (1987) Cell 48:703-12), myosin light chain-2 gene control regionwhich is active in skeletal muscle (Sani, (1985) Nature 314.283-6), andgonadotropic releasing hormone gene control region which is active inthe hypothalamus (Mason, et al. (1986) Science 234:1372-8).

Other promoters which may be used in the practice of the inventioninclude promoters which are preferentially activated in dividing cells,promoters which respond to a stimulus (e.g. steroid hormone receptor,retinoic acid receptor), tetracycline-regulated transcriptionalmodulators, cytomegalovirus immediate-early, retroviral LTR,metallothionein, SV-40, E1a, and MLP promoters.

Additional vector systems include the non-viral systems that facilitateintroduction of polynucleotide agents into a patient. For example, a DNAvector encoding a desired sequence can be introduced in vivo bylipofection. Synthetic cationic lipids designed to limit thedifficulties encountered with liposome-mediated transfection can be usedto prepare liposomes for in vivo transfection of a gene encoding amarker (Felgner, et. al. (1987) Proc. Natl. Acad Sci. USA 84:7413-7);see Mackey, et al. (1988) Proc. Natl. Acad. Sci. USA 85:8027-31; Ulmer,et al. (1993) Science 259:1745-8). The use of cationic lipids maypromote encapsulation of negatively charged nucleic acids, and alsopromote fusion with negatively charged cell membranes (Felgner andRingold, (1989) Nature 337:387-8). Particularly useful lipid compoundsand compositions for transfer of nucleic acids are described in WO95/18863 and WO 96/17823, and in U.S. Pat. No. 5,459,127. The use oflipofection to introduce exogenous genes into the specific organs invivo has certain practical advantages and directing transfection toparticular cell types would be particularly advantageous in a tissuewith cellular heterogeneity, for example, pancreas, liver, kidney, andthe brain. Lipids may be chemically coupled to other molecules for thepurpose of targeting. Targeted peptides, e.g., hormones orneurotransmitters, and proteins for example, antibodies, or non-peptidemolecules could be coupled to liposomes chemically. Other molecules arealso useful for facilitating transfection of a nucleic acid in vivo, forexample, a cationic oligopeptide (e.g. WO 95/21931), peptides derivedfrom DNA binding proteins (e.g. WO 96/25508), or a cationic polymer(e.g. WO 95/21931).

It is also possible to introduce a DNA vector in vivo as a naked DNAplasmid (see U.S. Pat. No. 5,693,622, U.S. Pat. No. 5,589,466 and U.S.Pat. No. 5,580,859). Naked DNA vectors for therapeutic purposes can beintroduced into the desired host cells by methods known in the art,e.g., transfection, electroporation, microinjection, transduction, cellfusion, DEAE dextran, calcium phosphate precipitation, use of a genegun, or use of a DNA vector transporter (see, e.g., Wilson, et al.(1992) J. Biol. Chem. 267:963-7; Wu and Wu, (1988) J. Biol. Chem.263:14621-4; Hartmut, et al. Canadian Patent Application No. 2,012,311,filed Mar. 15, 1990; Williams, et al (1991). Proc. Natl. Acad. Sci. USA88:2726-30). Receptor-mediated DNA delivery approaches can also be used(Curiel, et al. (1992) Hum. Gene Ther. 3:147-54; Wu and Wu, (1987) J.Biol. Chem. 262:4429-32).

The present invention also provides biologically compatible, cartilagedegradation-inhibiting compositions comprising an effective amount ofone or more compounds identified as TARGET inhibitors, and/or theexpression-inhibiting agents as described hereinabove.

A biologically compatible composition is a composition, that may besolid, liquid, gel, or other form, in which the compound,polynucleotide, vector, and antibody of the invention is maintained inan active form, e.g., in a form able to effect a biological activity.For example, a compound of the invention would have inverse agonist orantagonist activity on the TARGET; a nucleic acid would be able toreplicate, translate a message, or hybridize to a complementary mRNA ofa TARGET; a vector would be able to transfect a TARGET cell andexpression the antisense, antibody, ribozyme or siRNA as describedhereinabove; an antibody would bind a TARGET polypeptide domain.

A preferred biologically compatible composition is an aqueous solutionthat is buffered using, e.g., Tris, phosphate, or HEPES buffer,containing salt ions. Usually the concentration of salt ions will besimilar to physiological levels. Biologically compatible solutions mayinclude stabilizing agents and preservatives. In a more preferredembodiment, the biocompatible composition is a pharmaceuticallyacceptable composition. Such compositions can be formulated foradministration by topical, oral, parenteral, intranasal, subcutaneous,and intraocular, routes. Parenteral administration is meant to includeintravenous injection, intramuscular injection, intraarterial injectionor infusion techniques. The composition may be administered parenterallyin dosage unit formulations containing standard, well-known non-toxicphysiologically acceptable carriers, adjuvants and vehicles as desired.

A particularly preferred embodiment of the present composition inventionis a cartilage formation-enhancing pharmaceutical composition comprisinga therapeutically effective amount of an expression-inhibiting agent asdescribed hereinabove, in admixture with a pharmaceutically acceptablecarrier. Another preferred embodiment is a pharmaceutical compositionfor the treatment or prevention of a condition involving inflammation orcartilage and/or ECM degradation, or a susceptibility to the condition,comprising an effective cartilage formation-enhancing amount of a TARGETantagonist or inverse agonist, its pharmaceutically acceptable salts,hydrates, solvates, or prodrugs thereof in admixture with apharmaceutically acceptable carrier. In one aspect the conditioninvolves a systemic or local decrease in mean cartilage thickness.

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient. Pharmaceutical compositions for oral usecan be prepared by combining active compounds with solid excipient,optionally grinding a resulting mixture, and processing the mixture ofgranules, after adding suitable auxiliaries, if desired, to obtaintablets or dragee cores. Suitable excipients are carbohydrate or proteinfillers, such as sugars, including lactose, sucrose, mannitol, orsorbitol; starch from corn, wheat, rice, potato, or other plants;cellulose, such as methyl cellulose, hydroxypropylmethyl-cellulose, orsodium carboxymethyl-cellulose; gums including arabic and tragacanth;and proteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate. Dragee cores may be used in conjunction with suitablecoatings, such as concentrated sugar solutions, which may also containgum arabic, talc, polyvinyl-pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for product identification or to characterizethe quantity of active compound, i.e., dosage.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Preferred sterile injectable preparations can be a solution orsuspension in a non-toxic parenterally acceptable solvent or diluent.Examples of pharmaceutically acceptable carriers are saline, bufferedsaline, isotonic saline (e.g. monosodium or disodium phosphate, sodium,potassium; calcium or magnesium chloride, or mixtures of such salts),Ringer's solution, dextrose, water, sterile water, glycerol, ethanol,and combinations thereof 1,3-butanediol and sterile fixed oils areconveniently employed as solvents or suspending media. Any bland fixedoil can be employed including synthetic mono- or di-glycerides. Fattyacids such as oleic acid also find use in the preparation ofinjectables.

The compounds or compositions of the invention may be combined foradministration with or embedded in polymeric carrier(s), biodegradableor biomimetic matrices or in a scaffold. The carrier, matrix or scaffoldmay be of any material that will allow composition to be incorporatedand expressed and will be compatible with the addition of cells or inthe presence of cells. Preferably, the carrier matrix or scaffold ispredominantly non-immunogenic and is biodegradable. Examples ofbiodegradable materials include, but are not limited to, polyglycolicacid (PGA), polylactic acid (PLA), hyaluronic acid, catgut suturematerial, gelatin, cellulose, nitrocellulose, collagen, albumin, fibrin,alginate, cotton, or other naturally-occurring biodegradable materials.It may be preferable to sterilize the matrix or scaffold material priorto administration or implantation, e.g., by treatment with ethyleneoxide or by gamma irradiation or irradiation with an electron beam. Inaddition, a number of other materials may be used to form the scaffoldor framework structure, including but not limited to: nylon(polyamides), dacron (polyesters), polystyrene, polypropylene,polyacrylates, polyvinyl compounds (e.g., polyvinylchloride),polycarbonate (PVC), polytetrafluorethylene (PTFE, teflon), thermanox(TPX), polymers of hydroxy acids such as polylactic acid (PLA),polyglycolic acid (PGA), and polylactic acid-glycolic acid (PLGA),polyorthoesters, polyanhydrides, polyphosphazenes, and a variety ofpolyhydroxyalkanoates, and combinations thereof. Matrices suitableinclude a polymeric mesh or sponge and a polymeric hydrogel. In thepreferred embodiment, the matrix is biodegradable over a time period ofless than a year, more preferably less than six months, most preferablyover two to ten weeks. The polymer composition, as well as method ofmanufacture, can be used to determine the rate of degradation. Forexample, mixing increasing amounts of polylactic acid with polyglycolicacid decreases the degradation time. Meshes of polyglycolic acid thatcan be used can be obtained commercially, for instance, from surgicalsupply companies (e.g., Ethicon, N.J). A hydrogel is defined as asubstance formed when an organic polymer (natural or synthetic) iscross-linked via covalent, ionic, or hydrogen bonds to create athree-dimensional open-lattice structure which entraps water moleculesto form a gel. In general, these polymers are at least partially solublein aqueous solutions, such as water, buffered salt solutions, or aqueousalcohol solutions, that have charged side groups, or a monovalent ionicsalt thereof. The composition medium may be a hydrogel, which isprepared from any biocompatible or non-cytotoxic homo- orhetero-polymer, such as a hydrophilic polyacrylic acid polymer that canact as a drug absorbing sponge. Certain of them, such as, in particular,those obtained from ethylene and/or propylene oxide are commerciallyavailable. A hydrogel can be deposited directly onto the surface of thetissue to be treated, for example during surgical intervention.

Embodiments of pharmaceutical compositions of the present inventioncomprise a replication defective recombinant viral vector encoding thepolynucleotide inhibitory agent of the present invention and atransfection enhancer, such as poloxamer. An example of a poloxamer isPoloxamer 407, which is commercially available (BASF, Parsippany, N.J.)and is a non-toxic, biocompatible polyol. A poloxamer impregnated withrecombinant viruses may be deposited directly on the surface of thetissue to be treated, for example during a surgical intervention.Poloxamer possesses essentially the same advantages as hydrogel whilehaving a lower viscosity.

The active expression-inhibiting agents may also be entrapped inmicrocapsules prepared, for example, by interfacial polymerization, forexample, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(1980) 16th edition, Osol, A. Ed.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi-permeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™. (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated antibodiesremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for stabilization depending on the mechanisminvolved. For example, if the aggregation mechanism is discovered to beintermolecular S—S bond formation through thio-disulfide interchange,stabilization may be achieved by modifying sulfhydryl residues,lyophilizing from acidic solutions, controlling moisture content, usingappropriate additives, and developing specific polymer matrixcompositions.

As defined above, therapeutically effective dose means that amount ofprotein, polynucleotide, peptide, or its antibodies, agonists orantagonists, which ameliorate the symptoms or condition. Therapeuticefficacy and toxicity of such compounds can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., ED₅₀ (the dose therapeutically effective in 50% of the population)and LD₅₀ (the dose lethal to 50% of the population). The dose ratio oftoxic to therapeutic effects is the therapeutic index, and it can beexpressed as the ratio, LD₅₀/ED₅₀. Pharmaceutical compositions thatexhibit large therapeutic indices are preferred. The data obtained fromcell culture assays and animal studies are used in formulating a rangeof dosage for human use. The dosage of such compounds lies preferablywithin a range of circulating concentrations that include the ED₅₀ withlittle or no toxicity. The dosage varies within this range dependingupon the dosage form employed, sensitivity of the patient, and the routeof administration.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays or in animal models, usuallymice, rabbits, dogs, or pigs. The animal model is also used to achieve adesirable concentration range and route of administration. Suchinformation can then be used to determine useful doses and routes foradministration in humans. The exact dosage is chosen by the individualphysician in view of the patient to be treated. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Additional factors which maybe taken into account include the severity of the disease state, age,weight and gender of the patient; diet, desired duration of treatment,method of administration, time and frequency of administration, drugcombination(s), reaction sensitivities, and tolerance/response totherapy. Long acting pharmaceutical compositions might be administeredevery 3 to 4 days, every week, or once every two weeks depending onhalf-life and clearance rate of the particular formulation.

The pharmaceutical compositions according to this invention may beadministered to a subject by a variety of methods. They may be addeddirectly to TARGET tissues, complexed with cationic lipids, packagedwithin liposomes, or delivered to TARGET cells by other methods known inthe art. Localized administration to the desired tissues may be done bydirect injection, transdermal absorption, catheter, infusion pump orstent. The DNA, DNA/vehicle complexes, or the recombinant virusparticles are locally administered to the site of treatment. Alternativeroutes of delivery include, but are not limited to, intravenousinjection, intramuscular injection, subcutaneous injection, aerosolinhalation, oral (tablet or pill form), topical, systemic, ocular,intraperitoneal and/or intrathecal delivery. Examples of ribozymedelivery and administration are provided in Sullivan et al. WO 94/02595.

Antibodies according to the invention may be delivered as a bolus only,infused over time or both administered as a bolus and infused over time.Those skilled in the art may employ different formulations forpolynucleotides than for proteins. Similarly, delivery ofpolynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

As discussed hereinabove, recombinant viruses may be used to introduceDNA encoding polynucleotide agents useful in the present invention.Recombinant viruses according to the invention are generally formulatedand administered in the form of doses of between about 10⁴ and about10¹⁴ pfu. In the case of AAVs and adenoviruses, doses of from about 10⁶to about 10¹¹ pfu are preferably used. The term pfu (“plaque-formingunit”) corresponds to the infective power of a suspension of virions andis determined by infecting an appropriate cell culture and measuring thenumber of plaques formed. The techniques for determining the pfu titreof a viral solution are well documented in the prior art.

The present invention also provides methods of enhancing cartilageformation, which comprise the administration to said subject atherapeutically effective amount of an expression-inhibiting agent ofthe invention. A further aspect of the invention relates to a method oftreating or preventing a disease involving chondrocyte anabolicstimulation, comprising administering to said subject a cartilageformation-enhancing pharmaceutical composition as described herein.

Examples of diseases involving degradation of ECM that are treatableusing the means and methods of the present invention include, but arenot limited to psoriatic arthritis, juvenile arthritis, early arthritis,reactive arthritis, osteoarthritis, ankylosing spondylitis.osteoporosis, muskulo skeletal diseases such as tendinitis andperiodontal disease, cancer metastasis, airway diseases (COPD, asthma),renal and liver fibrosis, cardio-vascular diseases such asatherosclerosis and heart failure, and neurological diseases such asneuroinflammation and multiple sclerosis.

Examples of diseases involving degradation of cartilage that aretreatable using the means and methods of the present invention include,but are not limited to osteoarthritis, rheumatoid arthritis, psoriaticarthritis, juvenile rheumatoid arthritis, gouty arthritis, septic orinfectious arthritis, reactive arthritis, reflex sympathetic dystrophy,algodystrophy, Tietze syndrome or costal chondritis, fibromyalgia,osteochondritis, neurogenic or neuropathic arthritis, arthropathy,endemic forms of arthritis like osteoarthritis deformans endemica,Mseleni disease, and Handigodu disease; degeneration resulting fromfibromyalgia, systemic lupus erythematosus, scleroderma, and ankylosingspondylitis. Furthermore, people suffering from congenital cartilagemalformations, including hereditary chondrolysis, chondrodysplasias andpseudoachondrodysplasias, are likely to benefit from programs thatresult in anabolic stimulation of chondrocytes, and these diseasestherefore may also be treated by using the methods and means of thepresent invention. Non-limiting examples of congenital cartilagemalformation related diseases are microtia, anotia, and metaphysealchondrodysplasia.

In addition, as the identified targets do also inhibit IL-1 signaltransduction, inhibitors of these targets could be of use in thetreatment of inflammatory diseases. Examples of diseases involvinginflammation that are treatable using the means and methods of thepresent invention include but are not limited to allergic airwaysdisease (e.g. asthma, rhinitis), autoimmune diseases, transplantrejection, Crohn's disease, rheumatoid arthritis, psoriasis, juvenileidiopathic arthritis, colitis, and inflammatory bowel diseases.

In one aspect the present invention provides methods of preventingand/or treating disorders involving inflammation, ECM degradation and/orcartilage degradation, said methods comprising administering to asubject a therapeutically effective amount of an agent as disclosedherein. In a particular embodiment, the agent is selected from anexpression-inhibiting agent and an antibody. In a particular embodimentthe disorder is selected from osteoarthritis, rheumatoid arthritis,allergic airways disease (e.g. asthma, rhinitis), and autoimmunediseases. In a particular embodiment the disorder is osteoarthritis.

The invention also relates to the use of an agent as described above forthe preparation of a medicament for treating or preventing a diseaseinvolving inflammation, ECM degradation and/or cartilage degradation. Ina particular embodiment, the agent is selected from anexpression-inhibiting agent and an antibody. In a particular embodimentof the present invention the disease is selected from osteoarthritis,rheumatoid arthritis, allergic airways disease (e.g. asthma, rhinitis),and autoimmune diseases. In a particular embodiment the disorder isosteoarthritis.

The present invention also provides a method of treating and/orpreventing a disease involving inflammation, ECM degradation and/orcartilage degradation said method comprising administering, to a subjectsuffering from, or susceptible to, a disease involving cartilagedegradation, a pharmaceutical composition or compound as describedherein, particularly a therapeutically effective amount of an agentwhich inhibits the expression or activity of a TARGET as identifiedherein. In a particular embodiment the disorder is selected fromosteoarthritis, rheumatoid arthritis, allergic airways disease (e.g.asthma, rhinitis), and autoimmune diseases. In a particular embodimentthe disorder is osteoarthritis.

The invention also relates to an agent or a pharmaceutical compositionas described above for use in the treatment and/or prevention of adisease involving inflammation, ECM degradation and/or cartilagedegradation. In a particular embodiment the disorder is selected fromosteoarthritis, rheumatoid arthritis, allergic airways disease (e.g.asthma, rhinitis), and autoimmune diseases. In a particular embodimentthe disorder is osteoarthritis.

Administration of the agent or pharmaceutical composition of the presentinvention to the subject patient includes both self-administration andadministration by another person. The patient may be in need oftreatment for an existing disease or medical condition, or may desireprophylactic treatment to prevent or reduce the risk for diseases andmedical conditions characterized by cartilage degradation. The agent ofthe present invention may be delivered to the subject patient orally,transdermally, via inhalation, injection, nasally, rectally or via asustained release formulation.

Still another aspect of the invention relates to a method for diagnosinga pathological condition involving inflammation, ECM degradation and/orcartilage degradation, comprising determining the amount of apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 22-42 in a biological sample, and comparing theamount with the amount of the polypeptide in a healthy subject, whereinan increase of the amount of polypeptide compared to the healthy subjectis indicative of the presence of the pathological condition. In aparticular embodiment the disorder is selected from osteoarthritis,rheumatoid arthritis, allergic airways disease (e.g. asthma, rhinitis),and autoimmune diseases. In a particular embodiment the disorder isosteoarthritis.

Still another aspect of the invention relates to a method for diagnosinga pathological condition involving inflammation, ECM degradation and/orcartilage degradation, comprising determining the activity of apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 22-42 in a biological sample, and comparing theactivity with the activity of the polypeptide in a healthy subject,wherein an increase of the activity of polypeptide compared to thehealthy subject is indicative of the presence of the pathologicalcondition. Clearly, the activity and/or expression levels of the targetgenes as disclosed herein may have an effect on anabolic stimulation ofchondrocytes. It remains to be determined to what level the activityshould be elevated to diagnose for the disease. However, by comparinglevels found in patients, individuals without symptoms and clearlyhealthy individuals the skilled person may easily determine theserelevant levels. Since the skilled person is now aware whichpolypeptides should be monitored, the present invention provides noveltools for test assays for such diagnostics. A prominent disease that maybe controlled, checked and diagnosed by using the knowledge provided bythe present invention is osteoarthritis. In a particular embodiment thedisorder is selected from osteoarthritis, rheumatoid arthritis, allergicairways disease (e.g. asthma, rhinitis), and autoimmune diseases. In aparticular embodiment the disorder is osteoarthritis.

Still another aspect of the invention relates to a method for diagnosinga pathological condition involving inflammation, ECM degradation and/orcartilage degradation, comprising determining the nucleic acid sequenceof at least one of the genes of SEQ ID NO: 1-21 within the genomic DNAof a subject; comparing the sequence with the nucleic acid sequenceobtained from a database and/or a healthy subject; and identifying anydifference(s) related to the onset or prevalence of the pathologicalconditions disclosed herein. Such differences may be further checked inin vitro assays applying similar marker genes as disclosed herein. Suchassays will reveal the role of the gene or its encoded polypeptide inanabolic stimulation processes of chondrocytes. If such mutations areidentified this knowledge can be further exploited in test-kits fordiagnosis of similar diseases. In a particular embodiment the disorderis selected from osteoarthritis, rheumatoid arthritis, allergic airwaysdisease (e.g. asthma, rhinitis), and autoimmune diseases. In aparticular embodiment the disorder is osteoarthritis.

The polypeptides or the polynucleotides employed in the methods of thepresent invention may be free in solution, affixed to a solid support,borne on a cell surface, or located intracellularly. To perform themethods it is feasible to immobilize either the polypeptide of thepresent invention or the compound to facilitate separation of complexesfrom uncomplexed forms of the polypeptide, as well as to accommodateautomation of the assay. Interaction (e.g., binding of) of thepolypeptide of the present invention with a compound can be accomplishedin any vessel suitable for containing the reactants. Examples of suchvessels include microtitre plates, test tubes, and microcentrifugetubes. In one embodiment, a fusion protein can be provided which adds adomain that allows the polypeptide to be bound to a matrix. For example,the polypeptide of the present invention can be “His” tagged, andsubsequently adsorbed onto Ni-NTA microtitre plates, or ProtA fusionswith the polypeptides of the present invention can be adsorbed to IgG,which are then combined with the cell lysates (e.g., (³⁵S-labelled) andthe candidate compound, and the mixture incubated under conditionsfavorable for complex formation (e.g., at physiological conditions forsalt and pH). Following incubation, the plates are washed to remove anyunbound label, and the matrix is immobilized. The amount ofradioactivity can be determined directly, or in the supernatant afterdissociation of the complexes. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level of theprotein binding to the protein of the present invention quantitated fromthe gel using standard electrophoretic techniques.

Other techniques for immobilizing protein on matrices can also be usedin the method of identifying compounds. For example, either thepolypeptide of the present invention or the compound can be immobilizedutilizing conjugation of biotin and streptavidin. Biotinylated proteinmolecules of the present invention can be prepared from biotin-NHS(N-hydroxy-succinimide) using techniques well known in the art (e.g.,biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized inthe wells of streptavidin-coated 96 well plates (Pierce Chemical).Alternatively, antibodies reactive with the polypeptides of the presentinvention but which do not interfere with binding of the polypeptide tothe compound can be derivatized to the wells of the plate, and thepolypeptide of the present invention can be trapped in the wells byantibody conjugation. As described above, preparations of a labeledcandidate compound are incubated in the wells of the plate presentingthe polypeptide of the present invention, and the amount of complextrapped in the well can be quantitated.

Another embodiment of the present invention relates to a method for theuse of compounds which are able to ameliorate or to stabilize theproperties of chondrocytes, chondrocyte progenitors, or mesenchymal stemcells used for autologous cell or cartilage transplantation, eitherduring ex vivo culturing or after implantation. This amelioration can bethe result of a reduced level of ECM and/or cartilage degradingproteases in or around the implant. For example a candidate compound maybe administered locally via implantation of a membrane, sponge, or otherappropriate material onto which the desired molecule has been absorbedor encapsulated. Where an implantation device is used, the device may beimplanted into any suitable tissue or organ, and delivery of the desiredmolecule may be via diffusion, timed-release bolus, or continuousadministration.

The rate of ECM and/or cartilage degradation can typically be measuredby determining the deposition of cartilage, or cartilage components, orcartilage-containing extra-cellular matrix produced by the chondrocytes,in the medium. A cell-based ELISA, enzymatic assays, or other generaltechniques known in the art can be used to measure cartilage components,like the ones described in Walsh G., Proteins: Biotechnology andBiochemistry. John Wiley and Sons, 2001.

The invention is further illustrated in the following figures andexamples.

EXAMPLES Example 1: MMP13 Assay with NHACs

As described in the introduction, MMP13 has been identified in theosteoarthritis literature as one of the key players involved incatabolic events leading to the degradation of cartilage in the affectedjoints of osteoarthritic patients. Therefore, it was decided to initiatea functional genomics effort in order to identify factors that modulatethe expression of MMP13 in primary human chondrocytes activated with adisease relevant trigger. This assay is further referred to herein asthe “MMP13 assay”. The factors identified in this assay can be used asthe basis for the development of novel therapies for osteoarthritis.

The MMP13 assay that has been developed for the screening of theSilenceSelect collection has the following distinctive features:

-   -   The assay is run with primary human articular chondrocytes, but        with minimal adaptations, could be used for any other source of        primary chondrocytes, chondrocyte progenitors, or chondrocytic        cell lines, or any cell that is capable of producing MMP13.    -   The assay is run such that the primary human chondrocytes used        are in an environment that resembles their normal environment        (the cartilage matrix) as much as possible. The cells are grown        in a three-dimensional culture, avoiding cell adhesion to the        culture vessel.    -   The assay has been optimized for use with arrayed adenoviral        collections for functional genomics purposes.    -   With minimal adaptations, the assay can also be used to screen        compounds or compound collections    -   The assay can be run in high throughput mode.

The MMP13 assay with normal human articular chondrocytes (NHACs) isdescribed in more detail below. First, the protocol of the MMP13 ELISAthat has been developed is described in section 1. Then the culture andmaintenance of the primary chondrocytes is described in section 2. Thescreening protocol of the MMP13 assay with NHACs is described in section3. The composition and performance of the control plate is shown insection 4. An example of the performance of the screening of theSilenceSelect collection is given in section 5.

The MMP13 assay in NHACs has been screened against an arrayed collectionof 10946 different recombinant adenoviruses mediating the expression ofshRNA's in NHACs. These shRNA's cause a reduction in expression levelsof genes that contain homologous sequences by a mechanism known as RNAinterference (RNAi). The 10946 Ad-siRNA's contained in the arrayedcollection do target 6308 different transcripts. On average, everytranscript is targeted by 2 to 3 independent, different Ad-siRNA's. Theprinciple of the screening is illustrated in FIG. 1 and described indetail below.

1.1 MMP13 ELISA Protocol

Various antibodies and substrates were tested in order to develop anELISA with sufficient sensitivity to detect the MMP13 amounts producedby cultured NHACs. A 384-well format ELISA for measurement of MMP13 wasdeveloped. Various primary antibodies were tested, as well as variousELISA protocols leading to the following validated protocol formeasurement of MMP13 levels in 384 well plates. Black maxi sorb 384 wellplates (Nunc 460518) are coated with 5 μg/mL anti-MMP13 antibody MAB511(R&D Systems). The antibody is diluted in carbonate-bicarbonate coatingbuffer (1.59 g Na₂CO₃ (Sigma S-7795) and 2.93 g NaHCO₃ (Sigma S-5761) in1 L milliQ, adjusted to pH 9.6). After overnight incubation at 4° C.,plates are washed three times with 100 μL PBST (80 g NaCl, 2 g KCl(Sigma), 11.5 g Na₂HPO₄.7H₂O and 2 g KH₂PO₄ in 10 L milliQ; pH 7.4+0.05%Tween-20 (Sigma)) and blocked with 100 μL/well blocking buffer (5% nonfat dry milk in PBS). After 2 hrs of incubation at room temperature,plates are washed three times with 100 μL PBST. The PBST is then removedand 35 μL of sample is transferred to the ELISA plates. After overnightincubation at 4° C., plates are washed three times with PBST andincubated for 1 hr at 37° C. with 35 μL/well 1.5 mM APMA. A 10 mM APMAstock solution (prepared one day before) is stored at 4° C. (35.18 mgAPMA (Sigma A-9563) in 10 mL 0.1M NaOH (Merck 1.06469.1000)). The 10 mMAPMA stock solution is diluted to 1.5 mM in APMA buffer (10×APMA buffer:500 mM Tris (Roche 708976), 50 mM CaCl₂ (Sigma C-5080), 500 μM ZnCl₂(Sigma Z-0173), 1.5 M NaCl (Calbiochem 567441), 0.5% Brij35 (Sigma 430AG-6) and adjust to pH 7.0). After activation of MMP13 by APMA, platesare washed again three times with 100 μL PBST/well. OmniMMP Fluorescentsubstrate (Biomol P-126) is dissolved in OmniMMP buffer (10× OmniMMPbuffer: 500 mM Hepes (Sigma H4034), 100 mM CaCl₂ (Sigma C5080), 0.5%Brij35 (Sigma 430 AG-6; adjusted to pH 7.0) to a final concentration of0.01 mM. 35 μL of this substrate is added to each well. After anovernight incubation at 37° C., the active MMP13 in the sample hascleaved the substrate and released fluorescence. Readout is performed onthe EnVision (Perkin Elmer) using 320 nm excitation/405 nm emissionfilters.

1.2 Maintenance of the Primary Chondrocytes

Normal human articular chondrocytes (NHACs) passage 1, were acquiredfrom a commercial source (Cat No CC-2550, Cambrex Verviers, BE). Forevery experiment, a vial containing primary NHACs is thawed according tothe manufacturers protocol and cells are cultured in monolayer in a T80cell culture vessel in chondrocyte growth medium (CGM, Cat No CC3216,Cambrex, Verviers) under standard conditions (37° C., 5% CO₂). When thisculture reaches confluence, the cells are trypsinized according to themanufacturers protocol (using reagent pack Cat No CC-3233, Cambrex,Verviers, BE) and transferred to a new T175 culture vessel (1×10E+05cells/T175 flask). When these cultures reached confluence, cells weretrypsinized and subjected to the MMP13 assay. Therefore, the cells usedfor the MMP13 assay were only subcultured for 2 passages after thawing.

1.3. Screening Procedure

The optimal screening protocol is as follows: 96 well tissue cultureplates are coated with 50 μL of 1.5% of low melting point agaroseprepared in DMEM-F12 medium supplemented with 5% of FBS and a mixture of100 units/mL penicillin (Invitrogen) and 100 μg/mL streptomycin(Invitrogen) (this medium is further referred to as “assay medium”).NHACs were trypsinized and seeded in polypropylene 96 well plates (toavoid cell adhesion) at a density of 7500 cells/20 μL/well in DMEM-F12medium containing 5% of FBS and a mixture of penicillin andstreptomycin. The cells are then infected with an adenovirus mediatingthe expression of human Coxsackie/adenovirus receptor (hCAR) (at an MOI(multiplicity of infection, referring to the amount of viral particlesused per cell in the assay) of approximately 250) in order to facilitatethe subsequent infection with the Ad-siRNA viruses contained in theSilenceSelect collection. The human Coxsackie/adenovirus receptor is acoreceptor that is involved in the attachment of the adenovirus to thehuman cell membrane. Expression of this receptor in cells has beendescribed to facilitate subsequent infection with adenoviruses, forexample in T-cells (Schmidt et al., 2000). One day later, 12 μL Ad-siRNAvirus from each well of the SilenceSelect® collection (WO 03/020931),stored in 384 well plates (estimated titer of 1×10⁹ viral particles permL) was transferred with the aid of a 96/384 channel dispenser toindividual wells of the 96 well plates containing the NHACs. As theaverage titer of the adenoviral library is 1×10⁹ Virus Particles/mL,this represents an average MOI of about 1600. After addition of theAd-siRNA to the wells, an incubation step of one hour at 37° C. isperformed. The infection is performed in an arrayed fashion as each wellis infected with one individual type of Ad-siRNA from the SilenceSelect®collection. 40 μL of 0.8% low melting point agarose prepared in DMEMF12medium supplemented with 8% FBS and a mixture of penicillin andstreptomycin are then added to the wells. The content of the wells ismixed by pipetting up and down with a multichannel robot and 50 μL ofthe mixture is transferred to the 96 well plates coated with agarose. Tospeed up the solidification of the agarose, the plates are stored at 4°C. for approximately 15 minutes. 150 μL of assay medium is the added tothe wells. The infected NHACs are then incubated for four days to allowthe shRNA expression in the cells to reach sufficient levels and thegene silencing machinery in the cells to be fully primed and active.Four days after infection, the medium on the cells is refreshed withassay medium. One day later, the cells are triggered by addition ofassay medium containing 10 ng/mL recombinant human IL1b. Two days afteraddition of the trigger, supernatant is collected and stored at −80° C.until subjected to the MMP13 ELISA. 35 μL of the supernatant aresubjected to the MMP13 ELISA, which is performed in a 384 well plateformat. The protocol applied for the high-throughput compatible ELISA isdescribed in Example 1.1. The infection, medium replacement and mediumcollection steps were performed with a TECAN Freedom pipettor (TecanFreedom 200 equipped with TeMO96, TeMO384 and RoMa, Tecan AG,Switzerland).

1.4. Performance of the Control Plate

A 96 well control plate is generated to assess the quality of the assay.The control plates are produced in the same way as the SilenceSelect®collection. Multiple aliquots of this control plate are produced andstored at −80° C. For every screening run, a new aliquot of thescreening plate is thawed and tested to allow the performance of all thescreening runs to be compared. The composition of this plate is asfollows. Wells are filled with control viruses that are produced underthe same conditions as the SilenceSelect® adenoviral collection (WO03/020931). This control plate contains four sets of positive controlviruses (8 wells per positive control virus; P₁ (Ad5-RIT1_v5_KD), P₂(Ad5-SLC26A8_v2_KD), P₃ (Ad5-TRAF6_v4_KD), P₄ (Ad5-NFKBIA_KI)), arrangedin columns, interspaced with three sets of negative control viruses (16wells per negative control virus; N₁ (Ad5-Empty_KD), N₂(Ad5-M6PR_v1_KD), N₃ (Ad5-LacZ_KI)). The negative controls viruses aretested either in presence of IL1 (8 wells) or in the absence of IL1 (8wells). The positive control samples are selected either based onliterature information (P₃ (Ad5-TRAF6_v4_KD), P₄ (Ad5-NFKBIA_KI)) orbased on a preliminary screening of limited Ad-siRNA viruses (P₁(Ad5-RIT1_v5_KD), P₂ (Ad5-SLC26A8_v2_KD)). TRAF6 is known to be requiredfor the signal transduction downstream of IL1 (Cao et al., 1996), andover-expression of NFKBIA is known to inhibit NFKappaB, a transcriptionfactor known to be required for the expression of MMP13 (Karin, 1999).

The control plate is run in parallel with and under the same conditionsas the aliquot plates from the SilenceSelect® collection during thedifferent screening runs. A representative example of the performance ofthe control plate tested with the screening protocol described above isshown in FIG. 3. The data shown represent the levels of MMP13 producedby NHACs infected with the viruses contained in the control plate. Theaverage of the data obtained from the testing of 2 control plates areshown. The data indicate a clear upregulation of the MMP13 expression byIL1 treatment of the cells. The maximal MMP13 levels that are obtainedupon IL1 treatment of the cells is comparable for the 3 negativecontrols. All 4 positive controls reduced the IL1 response of the NHACs,with the strongest effects being observed for the P₃ (Ad5-TRAF6_v4_KD)and P₄ (Ad5-NFKBIA_KI) positive controls. These data confirm the goodquality of the screening.

Example 2: Screening of the SilenceSelect Collection

In total, 10946 viruses of the SilenceSelect collection are screened inthe MMP13 assay performed on primary NHACs cultured in agarose in 3Dformat, according to the protocol described in example 1.3. These 10946viruses cover 6308 transcripts, reflecting the redundancy built in theSilenceSelect® collection. This redundancy results in most transcriptsbeing targeted by multiple independent Ad-siRNAs contained within theSilenceSelect® collection. The data obtained during one of the screeningbatches are shown in FIG. 4. During this screening batch, 1536 Ad-siRNAscontained in 4 384 well plates of the SilenceSelect® collection arescreened in duplicate in the MMP13 assay. As mentioned in thedescription of the MMP13 assay on NHACs given in Example 1, the NHACcell culture, infection with Ad5-siRNAs and activation with IL1 isperformed in 96 well format. As such, every quadrant of a 384 well platecontained in the SilenceSelect® collection is used to infect two 96 wellplates containing NHACs in parallel. The supernatant obtained is thentransferred to a 384 well plate for the determination of the MMP13levels in the MMP13 ELISA described in Example 1.1. Transfer is plannedsuch that duplicate samples are measured on the same MMP13 ELISA plate.Data analysis is performed as follows:

First, for every 384 well plate individually, the data for all samplesare normalized and transformed into “normalized values” as follows. All384 datapoints are listed and the 5% highest and 5% lowest MMP13 signalsare removed from the list. Mean and standard deviation are thencalculated over all samples of this reduced list and based on these meanand standard deviation, the normalized data are calculated by applyingthe formula: normalized value sample A=[(raw MMP13 signal sampleA−mean)/(standard deviation)]. This transformation of the data per 384well plate allows comparison of samples of different screening batches.

Then a threshold value is determined to allow hit calling as follows.For every screening batch, two 96 well control plates are tested onparallel with the samples from the SilenceSelect® collection and everycontrol plate contains 3 negative control viruses (8 wells per negativecontrol virus). The average and standard deviation of the MMP13 signalfor the 3 negative controls contained in the control plates (3 times 16wells=48 wells in total) is calculated. Based on this data, variousthreshold values are expressed in relation to the standard deviation byapplying following formula: threshold value=[(average over the negativecontrols)−(“cutoff” times standard deviation over the negativecontrols)]. Various threshold values are then tested against the 48negative controls. A cutoff is selected such that it defines a thresholdvalue according to which less than 19% of the negative controls arelower than this threshold. Ad-siRNA viruses were nominated as primaryhits if both datapoint (expressed as normalized values) for theseAd-siRNAs scored below the selected cutoff value in the primary screen.

In FIG. 4, the data are shown that are obtained in one of the screeningbatches during the screening of the SilenceSelect® collection againstthe MMP13 assay on NHACs. In this screening batch, 1536 Ad-siRNAs weretested in duplicate. On the graph, the 2 datapoints (expressed in termsof normalized MMP13 level) obtained for an Ad-siRNA are plotted againsteach other. The cutoff determined for this screening batch (−1.8) isindicated with dotted lines. The data for Ad-siRNA's that generated 2datapoints that are below the selected cutoff and were nominated asprimary hits (36 Ad-siRNA's) are indicated as black dots, the data forAd-siRNA's that generated only one datapoint below the selected cutoff(100 Ad-siRNA's) are indicated as gray dots, and the data for thenon-hit Ad-siRNA's are indicated as white dots. The symmetry observedbetween the duplicate datapoints (the datapoints are concentrated arounda straight line) demonstrates the good quality and reproducibility ofthe screening.

The scheme shown in FIG. 6 displays the attrition obtained duringscreening and the selection of the hits analysed in more detail. Out ofthe 10946 Ad-siRNA's screened, 263 were identified as scoring induplicate. In addition, in order to extract maximal value out of theprimary screen, the list of viruses scoring only at one out of the 2datapoints was further analyzed in order to identify independent virusestargeting an identical transcript. The fact that 2 independentAd-siRNA's targeting the same gene through a different target sequenceare active in the MMP13 assay, albeit more weakly, is consideredvaluable information. This analysis added 106 hits to the primary hitlist, bringing the final number of primary hits to 369. These 369primary hits were further analyzed in a rescreen procedure described inExample 3.

Example 3: 3MOI Rescreen

3.1 Rescreening Protocol

In order to turn primary hits into confirmed hits, original hitAd-siRNA's are repropagated twice independently in order to produceindependent Ad-siRNA material. These repropagated viruses are thentested in the MMP13 ELISA on NHACs at 3 MOI's. The new virus material isscreened at 3 MOI's because the repropagation material could generatevirus material with a different titer as compared to the originalprimary hits contained in the SilenceSelect® collection. As 2repropagations are tested at 3 MOI's, 6 datapoints are generated forevery Ad-siRNA subjected to the ‘3MOI rescreen’. Only the hits thatscore for at least 1 MOI for both repropagation materials are consideredconfirmed hits.

For the repropagation step, the primary hits are picked from theSilenceSelect collection and repropagated in 96 well plates togetherwith positive and negative controls. A possible layout for 3 MOIrescreen runs is shown in FIG. 5A. Each repropagation plate has 40 wellscontaining hit viruses surrounded by 20 control wells. These controlwells contain either negative controls (N1(Ad5-Empty_KD); N2(Ad5-LacZ_KI); N3 (Ad5-M6PR_v1_KD); N4(Ad5-Empty_KD) or positivecontrols (P1 (Ad5-TRAF6_v4_KD); P2 (Ad5-RIT1_v5_KD); P6 (Ad5-Empty_KD);P7(Ad5-Empty_KD). For propagation, the crude lysates of the hitAd-siRNAs samples from the SilenceSelect® collection are picked andarranged together with controls in 96 well plates. As the containers ofcrude lysates are labeled with a barcode (Screenmates™, Matrixtechnologies), quality checks are performed on the plates. To propagatethe viruses, 2.25×104 PER.C6/E2A cells are seeded in 200 μL of DMEMcontaining 10% non-heat inactivated FCS in each well of a 96 well plateand incubated overnight at 39° C. in a humidified incubator at 10% CO₂.One μL of crude lysate from each hit Ad-siRNA, arranged in the 96 wellplates as indicated above, is then added to separate wells of PER.C6/E2Acells using a 96 well dispenser. After 7 to 10 days of incubation in ahumidified incubator at 10% CO₂, the re-propagation plates are frozen at−20° C., provided that complete CPE (cytopathic effect, indicating virusproduction has been complete) could be observed.

In a next step, the Ad-siRNAs contained in the repropagation plates aretested in the MMP13 assay on NHACs. For this test, the same protocol wasused as the one described in Example 2 for the primary screening, withthe only difference that 3 volumes of the repropagated viruses are usedto infect the chondrocytes subjected to the MMP13 assay: i.e. 8 μL, 12μL and 16 μL. All control wells except the 2 uninfected ones are thenactivated with IL1. In addition, the P6 and P7 controls are treated withstaurosporin (5 μM final concentration) and brefeldin A (0.5 μg/mL finalconcentration). These compounds are added one day before the addition ofthe IL1 trigger as well as one day later together with the addition ofthe IL1 trigger. The aim of the staurosporin and Brefeldin A addition tothe P6 and P7 control wells is to define the background MMP13 expressionlevels when secretion by the NHACs is completely blocked (brefeldin A)or when the cells are treated with a cytotoxic compound (staurosporin).The data of the 3 MOI rescreens are analyzed as follows.

First, for every plate, sample data are normalized using followingformula:Normalized MMP13 value sample A=[((raw MMP13 signal sample A)−(medianMMP13 signal over the negative controls))/(standard deviation over theMMP13 signal of the negative controls)].

The same basis is applied to generate the normalized value for thepositive controls:Normalized MMP13 value positive controls=[((Median MMP13 signal over thepositive controls))−(median MMP13 signal over the negativecontrols)/(standard deviation over the MMP13 signal of the negativecontrols)].

For the “3 MOI rescreens”, the positive controls are the wells treatedwith staurosporine and brefeldin A (P6 and P7), which are expected toreflect maximal inhibition of the IL1 induced MMP13 expression levels.Based on the normalized MMP13 values for the samples and the positivecontrols, the percentage inhibition is calculated for every sample byapplying following formula: percentage inhibition sample A=[(NormalizedMMP13 value sample A)/(Normalized MMP13 value positive controls)*100].

An example of the outcome of a 3 MOI rescreen is shown in FIG. 5B. Theexperiment shown represents the data obtained during the screening ofone 96 well repropagation plate (containing 40 primary hit AdsiRNA's aswell as the appropriate controls) in the NHAC MMP13 assay at 8 μl induplicate. Data are expressed as percentage inhibition of IL1-inducedMMP13 expression. The cutoff selected for hit calling is 40 percent(represented as a dotted line). The average percentage inhibition forthe P6 and P7 positive controls (100%) is represented as a blacktriangle, the average percentage inhibition for the P1 and P2 positivecontrols (48%) is represented as a cross and the average percentageinhibition for the N1, N2, N3 and N4 negative controls (−8.6%%) isrepresented as a gray triangle. Data for the Ad-siRNA's adhering to thehit calling criteria (2 datapoints are above the 40% cutoff) areindicated as white or grey circles, the data for the Ad-siRNA's notadhering to the hit calling criteria are indicated as squares.Duplicates are plotted next to each other (n=1 as a white circle, n=2 asa grey circle). For this repropagation plate and at this MOI, 11 out ofthe 40 primary hit Ad-siRNA's were confirmed.

The 3 MOI rescreen data obtained for all 13 preferred targets areindicated in Table 3. This table shows the TARGETS identified as activein the “3 MOI rescreen”.

TABLE 3 3MOI Additional Primary screen rescreen KD virus Synthetic siRNAADAM15 Active Active Active ADAMTS6 Active Active Active (2) ActiveGPR34 Active Active Active GPR43 Active Active Active EPHA5 ActiveActive Active MAP2K2 Active Active Active MC3R Active Active Active (4)Active MET Active Active Active Active STK32B Active Active Active (3)CSNK1G2 Active Active Active EDG4 Active Active Active MAP4K1 ActiveActive Active KCNN4 Active Active Active

A typical example of the outcome of a rescreen experiment is displayedin FIG. 6. In this rescreen procedure, 225 out of the 369 KD virus hitstested were confirmed. Out of these, 13 were selected for furthervalidation based on an “in silico” analysis in which various propertiesof candidate targets were analysed, as drugability, availability ofconsumables allowing screening of the target against small moleculecollections, level of conservation of the target sequence between humanand rodent orthologs.

3.2 Quality Control of Target Ad-siRNAs

The quality and identity of hit Ad-siRNAs are checked by PCR andsequencing as described further. Target Ad-siRNAs are propagated usingderivatives of PER.C6® cells (Crucell, Leiden, The Netherlands) in96-well plates, followed by sequencing the siRNAs encoded by the targetAd-siRNA viruses. PER.C6/E2A cells are seeded in 96 well plates at adensity of 40,000 cells/well in 180 μL of PER.C6/E2A medium. Cells arethen incubated overnight at 39° C. in a 10% CO₂ humidified incubator.One day later, cells are infected with 1 μL of crude cell lysate fromSilenceSelect® stocks containing target Ad-siRNAs. Cells are incubatedfurther at 34° C., 10% CO₂ until appearance of a cytopathic effect (asrevealed by the swelling and rounding up of the cells, typically at 7days post infection). The supernatant is collected, and the virus crudelysate is treated with proteinase K by adding to 4 μL Lysis buffer (1×Expand High Fidelity buffer with MgCl2 (Roche Molecular Biochemicals,Cat. No 1332465) supplemented with 1 mg/mL proteinase K (Roche MolecularBiochemicals, Cat No 745 723) and 0.45% Tween-20 (Roche MolecularBiochemicals, Cat No 1335465) to 12 μL crude lysate in sterile PCRtubes. These tubes are incubated at 55° C. for 2 hours followed by a 15minutes inactivation step at 95° C. For the PCR reaction, 1 μL lysate isadded to a PCR master mix composed of 5 μL 10× Expand High Fidelitybuffer with MgCl₂, 0.5 μL of dNTP mix (10 mM for each dNTP), 1 μL of“Forward primer” (10 mM stock, sequence: 5′ CCG TTT ACG TGG AGA CTC GCC3′ (SEQ. ID NO.: 59), 1 μL of “Reverse Primer” (10 mM stock, sequence:5′ CCC CCA CCT TAT ATA TAT TCT TTC C) (SEQ. ID NO.: 60), 0.2 μL ofExpand High Fidelity DNA polymerase (3.5 U/μL, Roche MolecularBiochemicals) and 41.3 μL of H₂O.

PCR is performed in a PE Biosystems GeneAmp PCR system 9700 as follows:the PCR mixture (50 μL in total) is incubated at 95° C. for 5 minutes;each cycle runs at 95° C. for 15 sec., 55° C. for 30 sec., 68° C. for 4minutes, and is repeated for 35 cycles. A final incubation at 68° C. isperformed for 7 minutes. 5 μL of the PCR mixture is mixed with 2 μL of6× gel loading buffer, loaded on a 0.8% agarose gel containing 0.5 μg/μLethidium bromide to resolve the amplification products. The size of theamplified fragments is estimated from a standard DNA ladder loaded onthe same gel. The expected size is approximately 500 bp. For sequencinganalysis, the siRNA constructs expressed by the target adenoviruses areamplified by PCR using primers complementary to vector sequencesflanking the SapI site of the pIPspAdapt6-U6 plasmid. The sequence ofthe PCR fragments is determined and compared with the expected sequence.All sequences are found to be identical to the expected sequence.

Example 4: On Target Validation

The strengths and advantages of the siRNA technology are well-recognizedand proved by the speed at which the technology has spread over thescientific community. Still the use of this technology asks for therequired skills and knowledge as 1) siRNAs may nonspecifically targetunrelated genes with only partial sequence-complementarity (off-targeteffects) and 2) the efficacy of siRNA's is difficult to predict (Pei etal., 2006). As such, it remains important to confirm the activity of aprimary hit identified in a KD virus screen in an independent setting.Two approaches were taken to this end. First, a set of additional KDviruses were produced which were designed to reduce the expression of acertain gene through different target sequences. Second, syntheticsiRNAs were purchased (Dharmacon) and used for target validation in thechondrocytic cell line SW1353.

4.1. On Target Analysis Using Additional KD Viruses

A set of KD viruses designed to reduce the expression of selectedpreferred hits through different target sequences were produced. TheseKD viruses were subsequently arrayed on 96 well plates together withpositive and negative control viruses. Identical controls were used asdescribed for the repropagation plates described for the 3MOI rescreen(Example 3). All additional different KD viruses targeting a particularhit were regrouped on a plate together with the original hit KD virusidentified for this target during the primary screen. These plates wererepropagated 2 times. Two copies of every repropagated plate was thentested in duplicate in 2 independent runs in the NHAC MMP13 assay asdescribed above for the primary screen, using an MOI of 12 μl. As such,8 datapoints were generated for every KD virus. The data were analysedas described above for the 3MOI rescreen (Example 3), converting thedata to % inhibition. KD viruses giving rise to a reduction of the MMP13levels of 35% for 4 out of the 8 datapoints generated were consideredvalidated in this assay. As such, 1 additional KD virus having thecapacity to reduce IL1_induced MMP13 expression levels in NHACs wasidentified for the following targets: ADAM15, GPR34, EPHA5, MAP2K2, MET,GPR43, 2 additional KD viruses were identified for ADAMTS6, 3 for STK32Band 4 for MC3R.

4.2. On Target Analysis Using Synthetic siRNA

The purpose of this experiment was to further validate adenoviral shRNAmediated KD effects on IL1 triggered MMP13 release using synthetic siRNAduplexes. The validation was done in a chondrosarcoma SW1353 cell linethat was previously shown to upregulate MMP13 expression in response toIL1 triggering.

4.2.1 Materials

Human chondrosarcoma SW1353 cells (Cat. No. HTB-94, ATCC) are grown in ahumidified 5% CO₂ incubator at 37° C. in DMEM (Cat. No. 41966-029,Gibco) supplemented with 10% heat-inactivated FBS (Hyclone) and 1×Penicillin/Streptomycin (Cat. No. 15140-122, Gibco) and subcultured (1:5split ratio) twice a week after trypsinization.

Ready-to-use gene silencing siRNA duplexes for genes of interest may beobtained from Dharmacon. siGENOME SMARTpool (or ON-TARGETplus set of 4)lyophilized stock reagents are reconstituted in 1× siRNA Buffer (Cat.No. B-002000-UB-015, Dharmacon) to achieve 2 μM concentrations andaliquots are stored at −20° C.

4.2.2 Procedure

siRNA duplexes are delivered into the SW1353 cells under optimizedconditions. SW1353 cells are plated in 96-well plates (Nunc) at 10 000cells/100 μL cell culture medium 24 hours prior to transfection. Cellsare transfected with the siRNA reagents (30 nM or 10 nM finalconcentration) using INTERFERin™ (Cat. No. 409-10,Polyplus-transfection) at a final concentration of 1 L/well, essentiallyaccording to the manufacturers instructions. In brief, the siRNA stockreagent is diluted in 50 μL serum-free OptiMEM (Cat. No. 51985-026,Gibco) and 1 μL INTERFERin™ reagent is added followed by immediatehomogenization for 10 sec and incubation at room temperature for 10-45minutes to allow INTERFERin™/siRNA complexes to form. During complexformation the medium on top of the SW1353 cells is replaced with 100 μLpre-warmed cell culture medium containing no antibiotics. 50 μL of theformed INTERFERin™/siRNA mix is then added to the cells and plates arereturned to the incubator at 37° C. and 5% CO₂.

After 72 hr, the medium on top of the cells is removed and replaced with100 μL pre-warmed culture medium containing 10 ng/mL recombinant humanIL1β (Cat. No. 200-01B, PeproTech) and 25 ng/mL recombinant human OSM(Cat. No. 295-0M, R&D Systems). Culture medium is DMEM/F12 (Cat. No.11320-074, Gibco) containing 5% heat-inactivated FBS and 1×Penicillin/Streptomycin.

24 hr after addition of the cytokines, the supernatant is collected andstored at −80° C. for later analysis of appropriate dilutions in theMMP13 ELISA (see example 1), MMP1 ELISA (as described in WO 2006/040357)and TIMP2 ELISA (as described in WO 2006/040357). As TIMP2 levels arenot influenced by the addition of the trigger, changes in amount ofTIMP2 secreted into the supernatant are used to assess the effect ofsiRNA gene-specific duplex transfection on cell viability/secretion.

The effect of siRNA duplex delivery for genes of interest on IL1/OSMmediated upregulation of MMP13 and MMP1 may be assessed in twoindependent experiments. In each experiment transfection of siRNAduplexes is performed in duplicate at two siRNA concentration (10 nM and30 nM). SMARTpool reagents targeting the human MMP1 (siGENOME SMARTpoolsiRNA MMP1), and one out of 4 selected individual siRNA duplexestargeting the human TRAF6 gene (siGENOME SMARTpool siRNA set of 4) andMMP13 gene (siGENOME SMARTpool set of 4) are used as positive controls.Cells transfected with the siCONTROL Non-Targeting siRNA pool or withthe GL2.2 duplex targeting luciferase at 30 nM and/or 10 nM are used asnegative controls. For MMP1 analysis, negative controls include bothsiRNA reagents at the two concentrations. For MMP13 analysis, negativecontrols include the non-targeting siRNA reagent at 30 nM and 10 nM andthe GL2.2 duplex at 10 nM. Additional wells transfected with the GL2.2control that are left unstimulated are included as non-triggeredcontrols. Positive, negative and non-triggered controls are included oneach 96-well plate. The collected supernatant of 4 different 96-wellplates are analysed on one 384-well ELISA and results may be analysed asfollows:

First, MMP13 and MMP1 numeric data for each well are recalculated aspercentage inhibition of the signal (% PIN) as follows:% PIN=100−((signal_(sample)−Av signal_(non-triggered control))/(Avsignal_(negative controls)−Av signal_(non-triggered control))*100)where,

-   -   Av signal_(non-triggered control) is the average of        non-triggered control of the 4 96-well plates all analysed on        the same 384 ELISA    -   Av signal_(negative controls) is the average of described        negative controls of 4 96-well plates all analysed on the same        384 ELISA

TIMP2 results are expressed as percentage inhibition of the signal (%PIN) according to the following formula:% PIN=100−((signal_(sample)−Av signal_(background))/(Avsignal_(negative controls)−Av signal_(background1))*100)where

-   -   Av signal_(background1) is the background signal of the TIMP2        ELISA i.e. signal obtained in absence of TIMP2    -   Av signal_(negative controls) is the average of all negative        controls of 4 96-well plates analysed on same 384 ELISA

Then, individual wells may be said to have a positive score if the % PINwas higher than 35% (for MMP1) or than 50% (MMP1). At these cutoffsettings none of the negative controls are found to have a positivescore. Results for transfections at 10 nM or 30 nM are given a scoringvalue of 1, only if both replicates are scoring above preset cutoffs. Inorder to assure that a drop in MMP1 or MMP13 expression is not caused byloss of cell viability, the TIMP2 results are taken into consideration.If TIMP2 signal is found to drop more than 35% for both replicates thenresult of MMP1 or MMP13 is not taken into account. For each screen afinal value is assigned to the target that was the sum of the scoringvalues at both siRNA duplex test concentrations after taking intoaccount the TIMP2 analysis. The effect of the siRNA duplex for a givengene is then believed to be a “true” effect if the sum of the finalvalues of both screens is higher or equal to 2. The target is consideredvalidated with synthetic siRNA if it scored as a true hit in either MMP1or MMP13. As such, following targets were considered validated using thesynthetic siRNA technology: ADAMTS6, MC3R, MET, CSNK1G2, EDG4, MAP4K1,KCNN4.

Taken together, the outcome of the on target validation exercise for the13 hits selected through the on target analysis is indicated in Table 3.Nine targets were validated through the identification of an additionalKD virus capable of recapitulating the effects of the original KD virushit identified in the primary screen and 7 (3 of which were validatedwith an additional KD virus) were validated using the synthetic siRNAtechnology. Expression of these validated targets in primary humanchondrocytes was further assessed (Example 5).

Example 5: Expression Analysis in Human Chondrocytes of the PreferredTargets

In order to be validated as preferred targets, the genes should beexpressed in chondrocytes. This may be assessed using quantitativereal-time PCR. Normal human chondrocytes from articular cartilage (NHAC)(Cambrex, Verviers, Belgium) are seeded into 9 cm culture dishes at 3million cells/dish in DMEM/F12 medium supplemented with 5% fetal calfserum (HighClone, Perbio, Erembodegem, Belgium). Two days later culturemedium is replaced by either DMEM/F12 medium supplemented with 5% fetalcalf serum, with or without 10 ng/mL of IL-13, or with Chondrocytedifferentiation medium (CDM, Cell Applications, San Diego, Calif.), withor without 10 ng/mL IL-13. Each condition is performed in duplicate.After incubation for 48 h the medium is removed and cells are processedfor RNA isolation using the RNeasy midi kit according to themanufacturer's instructions (Qiagen, Venlo Netherlands) and purified RNAis stored in aliquots at −20° C.

RNA is reverse transcribed to cDNA using the TaqMan® Gold RT kit(Applied Biosystems, Lennik, Belgium), according to the manufacturer'sinstructions (1× TaqMan® RT buffer, 5 mM MgCl2, 0.5 mM dNTP, 2.5 μMrandom hexamers, 10 U RNase inhibitor and 25 U multiscribe reversetranscriptase).

The cDNA is diluted 6-fold and 5 μL is used per PCR reaction in a 25 μLreaction for real-time QPCR in a ABI7000 instrument using either theSYBR® Green universal Mastermix or the TaqMan® Universal Mastermix (Bothfrom Applied Biosystems).

For primer development in SYBR® Green QPCR analysis, DNA sequences areextracted from the RefSeq sequence depository, or, if not available,from the GenBank collection. From these sequences, primer pairs suitablefor SYBR® Green QPCR are designed using the PrimerExpress software(Applied Biosystems). These primer pairs are checked for theirspecificity toward their target gene with the Blast software (NCBI,Entrez). Suitable primer pairs are ordered (Invitrogen) and used at 0.3μM concentration. The primer pairs are shown in FIG. 7.

Primer pair quality is monitored by melting point analysis, wherebypairs yielding more than one melting point are discarded, and bycomparison to a reference cDNA (Clontech Laboratories, Mountain View,Calif.) whereby the difference in melting point should not differ bymore than 1° C.

For the genes for which the SYBR® Green primer pair does not fulfill therequirements, a TaqMan® assay is used (Assay-on-demand, AppliedBiosystems).

Genes are considered as expressed if the average Ct value over allconditions was 37 or less.

Expression of the preferred targets in human articular chondrocytes asobtained by quantitative real-time PCR. Each Ct value is the average of8 independent RNA preparations of cells grown in 4 different conditions,as outlined in Example 5. The results are show in Table 5.

TABLE 5 Expression of the preferred targets in human articularchondrocytes Hit-ID 2nd hit-ID Target Gene Symbol average Ct valuesH54-001 MET 26.3 H54-016 STK32B 30.70 H54-023 GPR34 36.18 H54-024H54-025 GPR43 32.86 H54-044 MAP2K2 26.70 H54-058 ADAMTS6 32.24 H54-094KCNN4 32.11 H54-127 H54-305 ADAM15 26.04 H54-140 MAP4K1 29.77 H54-165MC3R 33.98 H54-257 EPHA5 30.76 H54-269 CSNK1G2 23.15 H54-340 EDG4 32.97

A Ct value of below 25 is considered high expression, between 25 and 30is considered good expression, between 30 and 35 is considered moderateexpression and above 35 is considered low expression. Therefore, goodexpression levels in primary chondrocytes could be demonstrated for mosttargets except for GPR34 (Ct=36), that displayed a low expression level.However, low expression levels may be observed for GPCRs and this lowexpression is not always predictive for low receptor activity

Example 6 Further Validation of EPHA5

Exemplary foregoing TARGET EPHA5 has been validated and confirmed bycompounds directed against EPHA5 which inhibit EPHA5 and also inhibitMMP13 activity in NHACs. The activity against EPHA5 may be tested usingthe assays as described in Examples 6.1 and 6.2 below, ability toinhibit MMP13 activity in NHACs may be tested as described in Example 7.

6.1 EPHA5 Inhibition—Biochemical Assay

Recombinant Epha5 (Millipore catalog number 14-639) is incubated with0.1 mg/mL Poly(Glu,Tyr)sodium salt (4:1), MW 20 000-50 000 (Sigmacatalog number P0275) in kinase reaction buffer (10 mM MOPS pH7.0, 1 mMDTT, 0.01% Trition-X100, 2.5 mM MnCl₂, 0.5 mM Na₃VO₄, 5 mMbeta-glycerolphosphate, 0.5 μM non-radioactive ATP, 0.25 μCi33P-gamma-ATP (Perkin Elmer, catalog number NEG602K) finalconcentrations with or without 5 μL containing test compound or vehicle(DMSO, 1% final concentration), in a total volume of 25 μL, in apolypropylene 96-well plate (Greiner, V-bottom). After 45 min at 30° C.,reactions are stopped by adding 25 μL/well of 150 mM phosphoric acid.All of the terminated kinase reaction is transferred to prewashed (75 mMphosphoric acid) 96 well filter plates (Perkin Elmer catalog number6005177) using a cell harvester (Perkin Elmer). Plates are washed 6times with 300 μL per well of a 75 mM phosphoric acid solution and thebottom of the plates is sealed. 40 μL/well of Microscint-20 is added,the top of the plates is sealed and readout is performed using theTopcount (Perkin Elmer). Kinase activity is calculated by subtractingcounts per minute (cpm) obtained in the presence of a positive controlinhibitor (10 μM staurosporine) from cpm obtained in the presence ofvehicle. The ability of a test compound to inhibit this activity isdetermined as:Percentage inhibition=((cpm determined for sample with test compoundpresent−cpm determined for sample with positive control inhibitor)divided by (cpm determined in the presence of vehicle−cpm determined forsample with positive control inhibitor))*100%.

Dose dilution series are prepared for the compounds enabling the testingof dose-response effects in the EPHA5 assay and the calculation of theIC₅₀ for each compound. Each compound is routinely tested atconcentration of 30 μM followed by a 1/3 serial dilution, 8 points (30μM-6.67 μM-2.22 μM-740 nM-247 nM-82 nM-27 nM-9 nM) in a finalconcentration of 1% DMSO. If potency of compound series is increased,more dilutions may be prepared and/or the top concentration may belowered (e.g. 5 μM, 1 μM).

6.2 EPHA5 Inhibition—Cell Assay

The assay principle is to determine inhibitor activity on the STAT3(Tyr705) phosphorylation level in HEK293 cells transiently transfectedwith STAT3 and EPHA5.

HEK293 are maintained in Dulbecco's Modified Eagle's Medium (DMEM)containing 10% heat inactivated fetal calf serum, 100 U/mL Penicillinand 100 μg/mL Streptomycin.

HEK293 at 70% confluency are collected by standard trysinisation.15,000,000 cells are transiently transfected with 6,250 ng ofpIPspAdapt6-STAT3, 3,750 ng of pIPspAdapt6-EPHA5, 4,000 ng ofpIPspAdapt6-eGFP and 11,000 ng of pBSK using 50 μL Jet-PEI (Polyplus) astransfection reagent per T175 cm² cell culture flask. The transfectedcells are seeded in T175 cm² cell culture flask. After overnightincubation at 37° C., 10% CO₂, transfection medium is removed and freshcell culture medium is carefully added to avoid cell detachment.

48 hour after transfection, medium is removed. Cells are detached withprewarmed cell dissociation solution (Sigma cat no. C5914). 60,000cells/40 μL of DMEM are seeded per well in 384-well plate. Then 10 μL ofcompound dilution (5×) in DMEM is added.

All compounds are tested in duplicate starting from 20 μM followed by a1/3 serial dilution, 8 points (20 μM-6.6 μM-2.2 μM-740 nM-250 nM-82nM-27 nM-9 nM) in a final concentration of 0.2% DMSO.

After 5 h incubation at 37° C., phosphor-Stat3(Tyr705) levels aredetermined using the AlphaScreen® SureFire® Phospho-STAT 3(Tyr705) AssayKit (From Perkin Elmer). Cells are lysed by addition of 15 μL of 1×lysis buffer. The plate is gently shaken for 20 min at room temperature,4 μL of lysate is transfered to the proxiplate, then 7 μL reactionbuffer/activation buffer mix containing alpha-beads are added and theplate is sealed with an aluminium seal, shaken for 5 minutes andincubated for 16 hours at RT in the dark.

The plates are read on the Envision using standard AlphaScreen settings.0.2% DMSO is used as a negative control (0% inhibition). The positiveand negative controls are used to calculate z′ and PIN values.Percentage inhibition=(1−((value determined for sample with testcompound present−value determined for sample with positive controlinhibitor) divided by (value determined in the presence of vehicle−valuedetermined for sample with positive control inhibitor)))*100%.

Example 7 MMP13 Inhibition Assay

Compounds identified as being active against one of the TARGETsidentified herein, may be directly tested in an MMP13 inhibition assay.

Normal human articular chondrocytes (NHAC, Lonza cat no CC-2550) aresuspended at a concentration of 1 600 000 cells per mL in medium (Gibco,DMEM:F12) containing 5% FBS and 1% pen/strep. 25 μL of the cellsuspension is then pooled with 25 μL 0.8% agarose in medium with 5%serum and 1% pen/strep and added to a well of a 96-well culture dishprecoated with 50 μL 1.5% agarose in medium with 5% serum and 1%pen/strep. After the agarose has set, 127.5 μL medium is added to eachwell. The cells are then treated with 7.5 μL compound at variousconcentrations for 1 hour followed by triggering with 15 μL 10 ng/mLIL-1b (final concentration: 1 ng/mL). 48 hours after triggering, thesupernatant is harvested and MMP13 secretion is measured. Each compoundis routinely tested at concentration of 30 μM followed by a 1/3 serialdilution, 8 points (30 μM-10 μM-3.33 μM-1.11 μM-370 nM-123 nM-41 nM-14nM) in a final concentration of 0.3% DMSO.

MMP13 activity is measured in an antibody capture activity assay. Forthis purpose, 384 well plates (NUNC, P6491, MaxiSorp black) are coatedwith 35 μL of a 1.5 μg/mL anti-human MMP13 antibody (R&D Systems,MAB511) solution for 24 hours at 4° C. After washing the wells 2 timeswith PBS+0.05% Tween, the remaining binding sites are blocked with 100μL 5% non-fat dry milk (Santa Cruz, sc-2325, Blotto) in PBS for 24 hoursat 4° C. Then, the wells are washed 2 times with PBS+0.05% Tween and 35μL culture supernatant containing MMP13 is added and incubated for 4hours at room temperature. Following this the wells are washed 2 timeswith PBS+0.05% Tween and MMP13 protein is then fully activated byaddition of 35 μL of a 1.5 mM 4-Aminophenylmercuric acetate (APMA)(Sigma, A9563) solution and incubation at 37° C. for 1 hour. Then, thewells are washed again with PBS+0.05% Tween and 35 μL MMP13 substrate(Biomol, P-126, OmniMMP fluorogenic substrate) is added. Afterincubation for 1 hour at 37° C. fluorescence of the converted substrateis measured in a Perkin Elmer Wallac EnVision 2102 Multilabel Reader(wavelength excitation: 320 nm, wavelength emission: 405 nm).Percentage inhibition=((fluorescence determined in the presence ofvehicle−fluorescence determined for sample with test compound present)divided by (fluorescence determined in the presence ofvehicle−fluorescence determined for sample without trigger))*100%.

The activity of compounds against GPR43 may be tested using the assaysas described Example 8 or 9 below

Example 8: GPR43 Phenotypic Assay—Neutrophil Migration Assay

8.1 Isolation of Neutrophils from Buffy Coats

A human buffy coat is diluted with an equal volume of ice cold DPBS(Invitrogen cat#14190169). 20 mL of diluted buffy coat down is gentlymixed with 4 mL of 140 mM citric acid, 200 mM sodium citrate and 220 mMDextrose in a 50 mL conical tube. 12 mL of a 6% Dextran/0.9% NaClsolution (W/V) in water is added gently and mixed by inverting the tubeup to 20 times. The total volume is then transferred to a fresh tube andincubated at room temperature for 1 hour for complete separation of thetwo phases. The yellow supernatant is then transferred to a new tube andcentrifuged at 1300 rpm for 12 minutes in a standard table topcentrifuge at 4° C. without brake. The supernatant is discarded and theremaining cell pellet is rapidly resuspended by pipetting up and down in12 mL of ice-cold water. After 20 seconds 4 mL of ice cold 0.6 M KCl isadded, mixed carefully and centrifuged at 1300 rpm for 12 minutes in astandard table top centrifuge at 4° C. without brake. This procedure isrepeated until no red blood cells remain. Finally the pellet isresuspended in 4 mL of DPBS and layered over 5 mL of Lymphoprep™(Nycomed Pharma, Cat#1114545) in a 15 mL tube. After centrifugation at1300 rpm for 12 minutes in a standard table top centrifuge at 4° C. withlow brake, the supernatant is removed and the cell pellet is resuspendedin 25 mL chemotaxis buffer (RPMI 1640 (Invitrogen, Cat#21875)supplemented with 10 mM of HEPES (Invitrogen, Cat#15630).

8.2 Migration Assay

The migration assay is performed in a Corning HTS transwell 96 permeablesupport system with 5.0 μM pore size polycarbonate membrane (CorningCat#3387). 180 μL of a cell suspension of 8.9×10⁶ cells/mL is added to20 μL of compound solution in chemotactic buffer in a polypropylene96-well V-bottom plate. The cells are incubated for 30 minutes with anintermediate resuspension after 15 minutes to prevent the cells fromsettling to the plate bottom. Following this the 70 μL cell suspensionis transferred to the upper compartment of the transwell system. Thereceiver well is filled with 200 μL chemotaxis buffer containingcompound and chemotactic agent. After incubation at 37° C. in 5% CO₂ for1 hour, the amount of cells that migrate to the receiver plate ismeasured by lysis and measuring ATP content of the lysate using ATP-lite(Promega, Cat#6016739) in a luminometer.

8.3 Data Analysis

Compound effects are expressed as percent inhibition using the formula:[(RLU in the presence of vehicle & chemotactic agent−RLU in presence ofcompound & chemotactic agent)/(RLU in the presence of vehicle &chemotactic agent−RLU in absence of chemotactic agent)]*100RLU=relative luminescence units.

Example 9: GPR43—Calcium Flux Assay

The activity against GPR43 may be tested using the assay as described inbelow

Clear bottom 384 well plates (Corning, ref. 3712) are coated with 25 μLpoly-D-Lysine (Sigma, ref. P-6407) diluted in PBS at a finalconcentration of 0.05 mg/mL and incubated for 30 minutes at 37° C.Polylysine is removed by 2 washes with PBS. Six thousand HEK293 cellsare seeded in 25 μL of DMEM complemented with 10% FBS, 10 μg/mLpuromycin and 1% penicillin/streptomycin. The plates are then incubatedfor 24 hours at 37° C./5% CO₂. Twenty-five μL of calcium 4 dye(Molecular devices, ref. R8141) are added to plates according tomanufacturer instructions and plates are incubated for 2 hours 15minutes at 37° C./5% CO₂. Ten μL of compound solutions in HBSS, 20 mMhepes are then added and plates are incubated at 37° C./5% CO₂ for 15minutes. Antagonist activity is measured by adding 10 μL of sodiumacetate (Sigma, ref. S2889) in HBSS, 20 mM hepes at the EC₈₀concentration.

Calcium signaling is measured using a Flex station3 (Molecular Devices)by recording fluorescence (excitation 485 nm, emission 525 nm) duringsixty seconds. Activity is determined as the ration between the maximalfluorescence of the calcium curves produced by the sodium acetate andthe basal fluorescence measured before sodium acetate addition.Percentages of inhibition are calculated using negative (vehicleaddition) and positive (sodium acetate at EC₁₀₀) controls on each plate.

Example 10: EDG4 Calcium Flux Assay

The activity of compounds against EDG4 may be tested using the assay asdescribed below

Ten thousand CHO-EDG4 cells per well are resuspended in 50 μL ofDMEM/F12 complemented with 10% FBS and 10 μg/mL Puromycin, then areseeded in clear bottom 384 well plates (Corning, ref. 3712). After 24hours incubation at 37° C./5% CO₂ plates are washed twice with 25 μL ofDMEM/F12 complemented with 0.1% fatty acid free BSA and then plates areincubated for 1 hour at 37° C./5% CO₂. Twenty-five μL of Fluo 4 DirectCa dye (Invitrogen, ref. F10473) diluted in HBSS containing calcium andmagnesium (Gibco ref. 14025) complemented with 20 mM HEPES (with further12 dilution compared to manufacturer instructions), and 5 mM Probenicid(Invitrogen, ref. P36400) are added to the plates. Plates are thenincubated for 1 hour at 37° C./5% CO₂. Ten μL of compound solutions inHBSS, 20 mM hepes, 0.1% fatty acid free BSA, 0.6% DMSO are then addedand plates are incubated at 37° C./5% CO₂ for 15 minutes. Antagonistactivity is measured by adding 10 μL of oleoyl-L-Lysophosphatidic acidsodium salt (LPA, Sigma, ref. L7260) in HBSS, 20 mM hepes, 0.1% fattyacid free BSA at the EC₈₀ concentration.

Calcium signaling is measured using a Flex station3 (Molecular Devices)by recording fluorescence (excitation 485 nm, emission 525 nm) for sixtyseconds. Activity is determined as the ratio between the maximalfluorescence of the calcium curve produced by the LPA and the basalfluorescence measured before LPA addition. Percentages of inhibition arecalculated using negative (vehicle addition) and positive (LPA at EC₁₀₀)controls on each plate.

The activity of compounds against CSNK1G2 may be tested using the assayas described in Example 11 or 12 below.

Example 11: CSNK1G2 Biochemical Assay

0.75 mU of recombinant CSNK1G2 (Millipore catalog number 14-712) isincubated with 0.1 mg/mL casein (Sigma catalog number C4765) in kinasereaction buffer (10 mM MOPS pH7.0, 0.01% Triton-X-100, 0.5 mM EDTA, 1 mMDTT, 0.5 mM Na₃VO₄, 5 mM beta-glycerophosphate, 10 mM MgCl₂, 0.5 μMnon-radioactive ATP, 0.25 μCi 33P-gamma-ATP (Perkin Elmer, catalognumber NEG602K) final concentrations with or without 5 μL containingtest compound or vehicle (DMSO, 1% final concentration), in a totalvolume of 25 μL, in a polypropylene 96-well plate (Greiner, V-bottom).After 45 min at 30° C., reactions are stopped by adding 25 μL/well of150 mM phosphoric acid. All of the terminated kinase reaction istransferred to prewashed (75 mM phosphoric acid) 96 well filter plates(Perkin Elmer catalog number 6005177) using a cell harvester (PerkinElmer). Plates are washed 6 times with 300 μL per well of a 75 mMphosphoric acid solution and the bottom of the plates is sealed. 40μL/well of Microscint-20 is added, the top of the plates is sealed andreadout is performed using the Topcount (Perkin Elmer).

Kinase activity is calculated by subtracting counts per minute (cpm)obtained in the presence of a positive control inhibitor (20 μMstaurosporine) from cpm obtained in the presence of vehicle. The abilityof a test compound to inhibit this activity is determined as:Percentage inhibition=((cpm determined for sample with test compoundpresent−cpm determined for sample with positive control inhibitor)divided by (cpm determined in the presence of vehicle−cpm determined forsample with positive control inhibitor))*100%.

Dose dilution series are prepared for the compounds enabling the testingof dose-response effects in the CSNK1G2 assay and the calculation of theIC₅₀ for each compound. Each compound is routinely tested atconcentration of 30 μM followed by a 1/3 serial dilution, 8 points (30μM-6.67 μM-2.22 μM-740 nM-247 nM-82 nM-27 nM-9 nM) in a finalconcentration of 1% DMSO. If potency of compound series is increased,more dilutions may be prepared and/or the top concentration may belowered (e.g. 5 μM, 1 μM).

Example 12: CSNK1G2 Cell Assay

In the Wnt-signaling assay a Wnt luciferase reporter construct istransfected together with expression vectors for LRP6 and CSNK1G2 intoSW1353 cells with the JetPEI transfection agent (Polyplus Transfection,Cat no 101-40). CSNK1G2 overexpression potentiates LRP6-inducedWnt-reporter activity. The CSNK1G2- and LRP6-mediated expression of theluciferase reporter gene is measured with a luciferase substrate.Compounds inhibiting CSNK1G2 activity will reduce reporter activity.

Day 1: a suspension of SW1353 cells is prepared (density: 30000cells/well/80 μL (375000 cells/mL)). In parallel, a DNA/transfectionagent mixture is prepared as follows.

The total amount of DNA to be added per well is diluted to 10 μL in aNaCl 150 mM solution. Typically, following amounts of DNA will betransfected per well: Wnt-luc reporter (20 ng), A010800-CSNK1G2-WT (20ng), pCS-myc-hLRP (10 ng). The total amount of JetPEI transfection agentneeded per well (0.32 μL) is diluted to 10 μL in a NaCl 150 mM solution.JetPEI solution and DNA solutions are then mixed yielding 20 μLDNA/transfection agent mix to be added per well. This solution isimmediately mixed, centrifuged and incubated for 30 min at RT. The cellsuspension is added dropwise to the DNA/transfection agent mix, andincubated at 37° C. for 2 hrs. The compound to be tested, diluted to 11μL, is then added to the wells containing the transfected cells using anautomated dispenser (Tecan aquarius) and the mixture is incubated for 16to 24 hours.

Day 2: The medium on top of the cells is removed and the luciferasesubstrate SteadyLite HTS (Perkin Elmer, CatNo 550-070303) is then addedto the white 96-well plates (50 μl/well). After an incubation of 30 minin the dark under continuous shaking, the readout is performed readoutwith a luminometer (Envision, Perkin Elmer).

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We claim:
 1. A method for identifying a compound that inhibits type IIcollagen degradation in extracellular matrix (ECM) and/or cartilage,comprising: (a) contacting a compound with a polypeptide comprising theamino acid sequence of SEQ ID NO: 29; (b) selecting a compound thatbinds said polypeptide; and (c) measuring the ability of the compoundselected in (b) to inhibit expression and/or activity of matrixmetalloproteinase 13 (MMP13).
 2. The method according to claim 1,wherein said polypeptide is in an in vitro cell-free preparation.
 3. Themethod according to claim 1, wherein said polypeptide is present in amammalian cell.
 4. The method of claim 1, wherein the method is used toidentify compounds that inhibit the catabolic process of chondrocytes.5. The method of claim 1, which comprises the steps of: c) contacting apopulation of mammalian cells expressing said polypeptide with one ormore compound that exhibits a binding affinity for said polypeptide; andd) identifying a compound that inhibits expression and/or activity ofMMP13 as a compound that inhibits type II collagen degradation in ECMand/or cartilage.
 6. The method of claim 1 or 5, wherein the compoundthat binds said polypeptide has a binding affinity to the polypeptide ofat least 10 micromolar.
 7. The method according to claim 1, whichadditionally comprises the step of comparing the compound to be testedto a control.
 8. The method according to claim 7, wherein said controlis where the polypeptide has not been contacted with said compound. 9.The method according to claim 5, which additionally comprises the stepof comparing the compound to a control, wherein said control is apopulation of mammalian cells that does not express said polypeptide.10. The method according to claim 1 or 5, wherein said compound isselected from the group consisting of compounds of a commerciallyavailable screening library and compounds having binding affinity for apolypeptide comprising the amino acid sequence of SEQ ID NO:
 29. 11. Themethod according to claim 1, wherein said compound is a peptide in aphage display library or an antibody fragment library.